DIETARY SUPPLEMENT COMPOSITION HAVING ENHANCED LSESr AND METHOD OF MAKING

ABSTRACT

A dietary supplement composition includes a shelf stable, supercritical CO2 fluid extracted, enhanced lipidosterolic extract of Serenoa repens (LSESr) having about an enrichment of lauric, myristic, oleic and linoleic free fatty acids to total free fatty acids greater than about 82.0% and a ratio of free fatty acids to total fatty acids greater than about 80.0%. A method of making the enhanced LSESr includes subjecting aged saw palmetto powder to supercritical CO2 extraction followed by first and second separations.

PRIORITY APPLICATION(S

This application is based upon provisional application Serial No.63/234,308, filed Aug. 18, 2021, the disclosure which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to the field of nutritional supplements, and moreparticularly, to a composition comprising a lipidosterolic extract ofSerenoa repens used to promote urinary and prostate function in men withenlarged prostates, or reduce androgenic hair loss in a patient, and amethod for extracting the lipid components and enhancing the free fattyacids from the Serenoa repens berries for prostate, skin, and hairhealth.

BACKGROUND OF THE INVENTION

Saw palmetto (Serenoa repens) is a shrubby palm that grows in thesoutheastern United States and is prolific on pine flatwoods inpeninsular Florida and southern Georgia. The saw palmetto grows a seedstalk (spadix) in late spring, which flowers and then develops a hardgreen fruit about the size of a small olive. The fruit ripens in thesummer, turning shades of yellow, orange and black. The ripe fruit isharvested by hand during the late summer and early fall.

Saw palmetto fruit (berries) have been ingested by Native Americans formany centuries. Early in the 20th century, naturopathic physicians inthe U.S. began prescribing a tea made from saw palmetto berries as amild diuretic and for genitourinary problems. It became a popular maletonic, but eventually the therapeutic value of the tea (a water extract)came into question, and in the early 1940’s it lost both its popularityand its inclusion in the National Formulary. Research in Europe in the1960’s led to the identification of the beneficial aspects of the lipidportion of the berry and its medically active components, including freefatty acids and sterols.

Saw palmetto was listed in the U.S. Pharmacopeia from 1900 to 1916, andthe National Formulary from 1925 to 1942. At various times from 1938 to1990, Food and Drug Acts and Amendments were passed to address theproblems of drug safety and effectiveness. Beginning in 1972, the FDAbegan evaluating over-the-counter drugs, including herbal preparations.In the U.S., however, it is permissible to sell herbal products only ifno claims or statements regarding their value in the prevention ortreatment of disease are made.

Saw palmetto berries are a common source for many modern nutraceuticalformulations. When the saw palmetto berries are initially harvested,they are usually placed into standard citrus boxes, which are thenloaded onto flatbed trailers, and transported by truck to dryingfacilities. The ripe berries contain approximately 66% water and aredried for several days at about 130-140° F., and after drying, may bestored for several years without deteriorating.

Dried berries are generally bagged and shipped to processing facilitieswhere they are ground into a powder. A therapeutically effective dosecan be prepared from a lipidosterolic extract of the dried berries. InEurope, ethanol or hexane solvents are commonly used to isolate thelipidosterolic extract from the berries, leaving a residue of the harshorganic chemicals remaining in the extract. These lipidosterolicextracts, such as the hexanic extracts, include fatty acids as variousesters and free fatty acids, sterols and long-chain (fatty) alcohols.The fatty acids include caproic, caprylic, capric, lauric, myristic,palmitic, palmitoleic, stearic, oleic, linoleic and linolenic acid.Sterols include beta-sitosterol, campesterol, stigmasterol andstigmastanol. Long-chain alcohols include octacosanol, hexacosanol,triacontanol and tetracosanol. Usually, this extract is formulated intoa convenient oral dosage form within a gelatin capsule.

Other techniques include the supercritical CO₂ extraction of sawpalmetto conducted at about 45° C. at about 220 bar to produce anextract SABALSELECT® that may be used without further purification, andincludes fatty acids, alcohols and sterols. These saw palmetto extractsvary widely in their final composition with free fatty acids ranging inconcentration from about 40% to below about 80%, methyl and ethyl estersranging from about 1.5% to 16.7%, and glycerides ranging from about 6.8%to about 52.2%.

The fact that the concentration of various fatty acids, esters, andglycerides can vary in a saw palmetto extract makes it challenging todetermine the effectiveness of any single saw palmetto extract, thusrequiring a biological assay of each manufactured lot produced as a sawpalmetto extract. To maintain continuous biological assays of eachmanufactured lot of saw palmetto extract to determine the impact onefficacy from changes in components, such as individual free fatty acidsand ratios of free fatty acids to total fatty acids is complex,inefficient and expensive. Better control over pre-extraction growth,harvesting and processing together with controlled extraction would beadvantageous that can be used to obtain a consistent, effectivestandardized lipid profile.

Lipidosterolic extracts of Serena repens (LSESr), may be sold alone,generally in 320 mg softgels, or in a variety of herbal formulations tosupport urinary and prostate health in men with prostate enlargement. Insome instances, saw palmetto extracts have also been used for reducingandrogenic hair loss in a patient. Certain saw palmetto extracts, suchas Permixon®, are regulated as a prescription drug in France andGermany, and are used by many patients for treatment of benign prostatichyperplasia (BPH), or enlarged prostate, but has drawbacks as a hexaneextract containing residual solvent.

In the United States, however, lipidosterolic extracts of Serenoa repensdried fruit are considered dietary supplements which may also providesome health benefits, including benefits associated with promotingurinary and prostate function, improve urinary flow and control, oraddress hair loss in a patient, including Androgenic Alopecia (AGA), orto promote healthy hair growth. The currently available saw palmettoproducts, such as Permixon®, are usually lipid extracts removed as thewhole lipid composition of the saw palmetto berry. Traditionally,extraction processes have not allowed for the formulation of a sawpalmetto extract that include a desired blend of the various lipidfractions, and therefore, it was challenging to custom formulate a LSESrcomposition to meet customer specifications and have optimal therapeuticvalue. Although some CO₂ extraction processes have yielded beneficialsaw palmetto extracts, further control to enhance the percentages andratios of compositional compounds, including different free fatty acidswith a reduction in esters and glycerides, and an extended shelf lifeand low peroxide value without added antioxidants and stabilizers, wouldbe beneficial for specific applications, such as prostate function, hairgrowth and health, skin care, such as acne remedies, and otherapplications.

SUMMARY OF THE INVENTION

In general, a method of producing an enhanced lipidosterolic extract ofSerenoa repens (LSESr) may comprise cryogenically milling at atemperature no greater than -20° C.entigrade, dried saw palmetto berrieshaving greater than about 10% moisture into a saw palmetto powder. In anexample, the saw palmetto berries may have a moisture from about 10% toabout 13%. The method includes ageing the saw palmetto powder for atleast 15 days and subjecting the aged saw palmetto powder tosupercritical CO₂ extraction at a first extraction pressure and firstextraction temperature to produce a CO₂ extract. The method furtherincludes fractionating the CO₂ extract at a first separation pressureand first separation temperature to produce a first fraction, followedby fractionating at a second separation pressure and second separationtemperature to produce a second fraction and an enhanced LSESr having aratio of free fatty acids to total fatty acids that is greater thanabout 80.0% and an enrichment of lauric, myristic, oleic and linoleicacids as free fatty acids to total free fatty acids that is greater thanabout 82.0%.

The enhanced LSESr has a balance of about 90.0% total fatty acids toabout 72% of free fatty acids. The enhanced LSESr has an enrichment oflauric, myristic, oleic and linoleic free fatty acids to total freefatty acids of about 82.0% to about 84.0%. The enhanced LSESr has aratio of free fatty acids to total fatty acids of about 80.0% to about82.0%. The supercritical CO₂ extraction may occur at an extractionpressure of about 495 bar to about 695 bar at a temperature of about 77to about 94° Cntigrade. The first fractionation may occur at about 126bar to about 350 bar at a temperature of about 35 to about 85°Cntigrade. The second fractionation may occur at about 30 bar to about80 bar at a temperature of about 12 to about 28° Cntigrade.

The method further includes cryogenically milling the saw palmettoberries into a ground saw palmetto powder having about 100% less thanabout 4 mesh and about 95% less than about 12 mesh, which may also occurat a temperature of about -50 to about -30° Cntigrade. The enhancedLSESr may have a peroxide value that is less than about 3 meq/kg andshelf stability of at least about 4 years without added antioxidants andstabilizers. The method may include ageing the saw palmetto powder forabout 15 to about 30 days. The first and second extracts may be blendedin various proportions to form the enhanced LSESr. The method mayinclude ageing the saw palmetto powder at a temperature from a mean ofabout 59 degrees to about 78° F., where the aging is in excess of about15 days.

In yet another example, a dietary supplement composition may comprise ashelf stable, supercritical CO₂ fluid extracted, enhanced lipidosterolicextract of Serenoa repens (LSESr) and include an enrichment of lauric,myristic, oleic and linoleic free fatty acids to total free fatty acidsgreater than about 82.0% and a ratio of free fatty acids to total fattyacids greater than about 80.0%.

The composition may be formulated into an oral dosage form. Thecomposition may be formulated into an oral dosage form and in aconvenient dosage capsule of about 160 mg b.i.d. to about 320 mg, orabout minimum 200 mg per dose of enhanced LSESr. The composition mayinclude less than about 0.2 percent w/w of saw palmetto sterols. Thecomposition may include a dry excipient comprising at least one ofsilicon dioxide, calcium silicate, calcium phosphate, magnesium oxide,magnesium carbonate, calcium carbonate, rice fiber and maltodextrin.

DESCRIPTION OF THE DRAWINGS

Other objects, features and advantages of the present invention willbecome apparent from the detailed description of the invention, whichfollows when considered in light of the accompanying drawings in which:

FIG. 1 is an example of a high level flowchart showing an example methodof producing the lipidosterolic extract of Serenoa repens (LSESr).

FIG. 2 is a table showing the 5α-Reductase Type 1 and Type 2 isoenzymeIC₅₀ pg/ml inhibition by the free fatty acids found in a saw palmettoextract.

FIG. 3 is another table showing the relative 5α-Reductase Type 1 andType 2 isoenzyme IC₅₀ µg/ml inhibition levels by the individualmolecules found in the saw palmetto extract as shown in FIG. 2 .

FIG. 4A is a lipid profile comparison among different samples (lots) ofexperimental LSESr compared to commercially available Permixon® andshowing how pre-extraction processing parameters and supercritical CO₂extraction parameters result in the more customized ratio of free fattyacids to total fatty acids such as Extraction Experiment Nos. 5 and 6 asLot Nos. 211022 and 211105 as examples of the enhanced LSESr of theinvention.

FIG. 4B is a table summarizing biological activity for specific samplesidentified in FIG. 4A and identifying the better profile of ExperimentNos. 5 and 6 as examples of the enhanced LSESr of the invention.

FIG. 5 shows a graph and bar chart for Inhibition Experiment 1 showingthe inhibition of 5α-Reductase 2 for two experimental extracts whencompared with commercially available Permixon^(®) that containspolyethylene glycol (PEG) in the formulation. The two experimentalsamples of LSESr produced from supercritical CO₂ extractions (U4602) and(U4868) contain pure extact and no PEG in Extraction Experiments 1 and2.

FIG. 6 is a graph for Inhibition Experiment 2 showing the inhibition of5α-Reductase Type 2 using the PEG-containing (pegylated) commerciallyavailable product Permixon^(®) compared to two samples of extractionexperiments as LSESr, e.g., one that is the pure extract (U4602-1;normal) and the other pegylated (U4602-PEG) .

FIG. 7A are graphs showing that 5α-Reductase Type 1 enzymes are presentin higher levels in the scalps of women with female pattern hair loss(FPHL), also known as androgenic alopecia (AGA), versus women withnormal scalps as shown in the left-hand graph. 5α-Reductase Type 2enzymes are also overexpressed in women with FPHL as shown in theright-hand graph.

FIG. 7B is a graph and bar chart showing the inhibition of 5α-ReductaseType 1 when compared with a sample from the supercritical CO₂ LSESr suchas from Extraction Experiments 5 and 6 and commercially availablePermixon®.

FIG. 7C is a graph and bar chart showing the inhibition of 5α-ReductaseType 2 enzyme when compared with a sample from the supercritical CO₂LSESr such as from Extraction Experiments 5 and 6 and commerciallyavailable Permixon®.

FIG. 7D is a bar chart showing the distribution of oral linolenic acidin guinea pigs and indicating how linolenic acid that may be enhancedsuch as in the enhanced LSESr of the current invention and is importantfor hair health and growth.

FIG. 8 are graphs showing a histogram of the cryogenic mill amperage inan example of the saw palmetto berry processing for producing theenhanced LSESr.

FIGS. 9A and 9B are graphs showing the normative test temperature indegrees Fahrenheit and test percent for relative humidity and showingthe aging impact on free fatty acids.

FIGS. 10A and 10B are charts corresponding to the graph and charts ofFIGS. 9A and 9B showing the temperature in degrees Fahrenheit andpercentage for relative humidity.

FIG. 11 is a graph showing the scatter plot of the five active freefatty acids as lauric, myristic, oleic, linoleic, and linolenic and theratio of free fatty acids to fatty acids relative to the mill and holdtime in days and showing how about 23 days gave the best ratio.

FIGS. 12A and 12B are bar charts showing the effect of processing withless than 10% moisture as below specifications (FIG. 12A) and atspecifications of about 10% to 12% moisture (FIG. 12B), and showing theaverage of the five active free fatty acids, and the ratio of free fattyacids to fatty acids.

FIG. 13 is a graph showing a scatter plot of the five active free fattyacids/fatty acids versus time between harvest and extraction.

FIG. 14 is a line chart showing the analysis of variance (ANOVA) for theratio of free fatty acids versus fatty acids on the five key free fattyacids per the mill and hold time.

FIG. 15 is a line chart showing the brackets hold time impact on theequal variances of free fatty acids/fatty acids.

FIGS. 16-20 are the analysis of variance line charts for the respectivepercentage of each of five actives as respective lauric, myristic,oleic, linoleic, and linolenic free fatty acids, and showing theparticular individual fatty acids and percentage of the free fatty acidsversus fatty acids.

FIGS. 21-26 are tables of the statistics and ranges for the respectivefive active fatty acids shown in the line charts of FIGS. 16-20 andshowing the percentage of free fatty acids versus fatty acids and postmill hold time in days.

FIG. 27 is a line graph showing the post milling hold time brackets forthe five active fatty acids and the ratio of free fatty acids versusfatty acids.

FIG. 28 is a line graph showing the test for equal variances for thefive active fatty acids and the ratio of free fatty acids versus fattyacids.

FIGS. 29-33 are line graphs showing the respective five active fattyacids weight fraction versus the post mill hold time for the fiveactives of FIGS. 16-20 .

FIG. 34 is a line graph similar to that shown in FIGS. 29-33 , butshowing the total fatty acids weight fraction versus the post mill holdtime.

FIG. 35 is a table showing the multiple regression for the total freefatty acids/fatty acids.

FIG. 36 are bar charts showing the multiple regression for the totalfree fatty acids/fatty acids.

FIG. 37 are graphs showing the multiple regression for the total freefatty acids/fatty acids and hold time and the effects, and showing howthe total ratio changes when settings are changed.

FIG. 38 are bar charts and scatter plots showing the multiple regressionsummary report for total free fatty acids/fatty acids and impact of xvariables.

FIG. 39 are scatter plots showing the multiple regression for total freefatty acids/fatty acids as a diagnostic report of residuals versusfitted values.

FIG. 40 are bar charts showing the multiple regression for total freefatty acids and incremental impact of x variables.

FIG. 41 are bar charts and scatter plots showing the multiple regressionfor total free fatty acids and impact of different variables.

FIG. 42 are graphs showing the multiple regression for total free fattyacids and how the total free fatty acids change if the settings of two xvariables are changed.

FIG. 43 are scatter plots showing the multiple regression for total freefatty acids and residuals versus fitted values.

FIG. 44 is a graph of a fitted line plot showing total free fatty acidsand lauric acid.

FIG. 45 is a graph showing a scatter plot of lauric acid/free fatty acidratio versus the percent oil in berries.

FIG. 46 is a graph similar to FIG. 45 , but showing a scatter plot ofthe lauric acid and fatty acid ratio versus the percent oil in berries.

FIG. 47 is a graph showing a scatter plot of the lauric acid/free fattyacid ratio versus hold time in days.

FIG. 48 is another graph of a scatter plot of the lauric acid and freefatty acid ratio versus the finish oil per biomass.

FIG. 49 is an analysis of variance line charts showing the free lauricacid per mill and hold.

FIG. 50 is another analysis of variance line charts showing the freefatty acid per mill and hold time.

FIG. 51 are bracket line charts showing the test for equal variances oflauric acid versus the hold time.

FIG. 52 are bracket line charts similar to FIG. 51 showing the test forequal variances of total free fatty acids versus the bracket hold time.

FIG. 53 is a line graph showing the analysis of variance for free fattyacids per mill and hold time.

FIG. 54 is another bracket line chart showing the test for equalvariances for the total free fatty acid versus the brackets hold time.

FIG. 55 is a high-level flowchart showing a method for producing theLSESr batches such as Experiments 5 and 6 as shown in FIGS. 4A and 4Busing supercritical CO₂ extraction to produce the enhanced LSESr of thecurrent invention.

FIG. 56 is a table of comparative data for high and low pressureextraction using the same raw material.

DETAILED DESCRIPTION

The present invention will now be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. This invention may, however, be embodied inmany different forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

A method of processing saw palmetto berries to form a lipidosterolicextract as a dietary supplement composition by supercritical fluidextraction using CO₂ under high pressure is illustrated in the highlevel flowchart of FIG. 1 shown generally at 100.

The LSESr of the current invention is an enhanced LSESr that is improvedover hexane solvent extracts such as commercially available Permixon®that contains residue of hexane and as it is environmentally friendlyprocess. For purposes of description, the LSESr of the current inventionwill be described as an enhanced LSESr and has a favorable andadvantageous 1) ratio of free fatty acids to total fatty acids; 2)higher enrichment of the free fatty acids of lauric, myristic, oleic,and linoleic as free fatty acids to total free fatty acids; and 3)specific free fatty acids tailored for specific end uses, such as moreenhanced linoleic and linolenic free fatty acids such as above 3.7% oftotal free fatty acids for hair health, and such as in uses forprostate, hair health, and skin care with other free fatty acids. It hasbeen found as explained further below that pre-extraction processing ofthe saw palmetto berry before the CO₂ extraction has an important rolein the final enhanced LSESr free fatty acid and fatty acid componentsthat gives the best support for prostate, hair, and skin uses.

For example, hair happens to have numerous androgen receptors and theenhanced LSESr is especially useful for inhibiting the activity of5α-Reductase-1 and 2 in certain individuals compared to the scalp ofnormal individuals, which has been seen to have a beneficial effect onhair health. The enhanced LSESr in an example, such as described belowwith reference to FIGS. 4A and 4B and the flowchart of FIG. 55 , has abetter profile not due to total higher fatty acids or higher free fattyacids or the level of important free fatty acids. The better profile isdriven by pre-extraction handling and CO₂ extraction parameters for ripeberries to achieve the right balance of free fatty acids to total(greater than about 80%), and at the same time, achieve a higherenrichment of the four important bioactive fatty acids (lauric,myristic, oleic, and linoleic) as a proportion of free fatty acids(greater than about 82.0%), thus delivering the most potent enrichmentof bioactive fatty acids. It has been found that the concentration oftotal fatty acids is less important than the ratio of free fatty acidsto total fatty acids and the optimized contribution of the fourbioactive free fatty acids as a percentage of total free fatty acids.

For example, enhanced berry processing and specific extraction andseparation pressure, temperatures and even times may be selected to forma final product as the enhanced LSESr of the current invention asdescribed above, which also provides for a peroxide value that is lessthan about 3 meq/kg and a shelf stability of at least 4 years withoutadded antioxidants and stabilizers. Generally, the enhanced LSESr is ashelf stable, supercritical CO₂ fluid extracted, enhanced lipidosterolicextract of Serenoa repens (LSESr), the LSESr having a ratio of freefatty acids to total fatty acids that is greater than about 80.0% and anenrichment of lauric, myristic, oleic and linoleic acids as free fattyacids to total free fatty acids that is greater than about 82.0%. In anexample, the enhanced LSESr may have a balance of about a preferred90.0% of total fatty acids to about 72.0% free fatty acids, and in aspecific example, about 88.9% to about 90.2% of total fatty acids toabout 72.4% to about 72.6%. The enhanced LSESr may have an enrichment oflauric, myristic, oleic and linoleic free fatty acids to total freefatty acids of about 82.0% to about 84.0%. The enhanced LSESr may have aratio of free fatty acids to total fatty acids of about 80.0% to about82.0%. These are non-limiting ranges and the values may range from about2 to 3% above and below these ranges.

The enhanced LSESr may be used in a composition formulated in atherapeutic amount to maintain and promote hair health and growth in ahuman in need thereof. In this example the composition is ashelf-stable, supercritical CO₂ fluid extracted, enhanced LSESr that hasa ratio of free fatty acids to total fatty acids that is greater thanabout 80.0% and an enrichment of lauric, myristic, oleic and linoleicacids as free fatty acids to total free fatty acids that is greater thanabout 82.0%, and an enrichment of linoleic and linolenic acids togetheris greater than about 3.7%. The composition may be formulated as atopical lotion to be applied to the hair. The composition may includeany one or more of zinc, vitamin D, rosemary oil and olive oil. Thecomposition may include up to about 5.0% of Minoxidil.

The enhanced LSESr is preferably not contained on a carrier such as PEG(polyethylene glycol) to stabilize the free fatty acids, as compared tothe commercially available Permixon®, which is on the PEG carrier. Inaddition to the four active free fatty acids listed above, a fifth aslinolenic free fatty acid has been found to be advantageous to help with5α-Reductase Type 1 as well as 5α-Reductase Type 2 as an inhibitor forhair treatment. The enhanced LSESr is produced using the inventivepreprocessing, extraction and separation parameters to deliver the mostactive form of the free fatty acids for prostate and hair health. Use ofpreprocessing and pre-extraction processing, such as specific grindingand blending, gives better plating and densifies the powder. Thefront-end processing and aging of berries and attendant time sequencingof the saw palmetto powder after grinding allows for the exposure of theinternal cell structure that helps the overall reaction and end product.These parameters include micromilling under cryogenic conditions andholding or ageing it.

Free fatty acids of the enhanced LSESr are better localized to theprostate, especially as a food source over glucose. The enhanced LSESrinhibits both the 5α-Reductase-1 and 2 enzymes that are especiallybeneficial for hair growth, and inhibits the 5α-Reductase-2 enzyme thatis especially beneficial for prostate health. The free fatty acids ofthe enhanced LSESr localize to tissues with high high androgen receptoractivity, like the prostate or the scalp, and can impact androgenreceptors like 5α-Reductase 1 and 2. The preprocessing parameters asdescribed below in greater detail help open up the berry matrix. Thefree fatty acids are better able to insert into the nuclear membranewith its attendant inhibitory effects on 5α-Reductase to preventconversion of testosterone to DHT. Normal and routine extraction of sawpalmetto berries produces usually about 70% free fatty acids or less,which is well below the percentage of free fatty acids in the enhancedLSESr of the current invention.

Androgens play an important role in prostate growth and development andin hair growth. Chief among these androgens are testosterone anddihydrotestosterone (DHT). The 5α-reductase (5αR) enzymes areresponsible for converting testosterone into the more potentdihydrotestosterone (DHT). There are three different types, or isoforms,of the 5α-reductase (5αR) enzymes: 5αR-1, 5aR-2, and 5αR-3. Theseisoforms can be found in various tissues and in varying amountsthroughout the body.

However, 5αR-2 in particular can become overexpressed in prostates ofaging men leading to prostate enlargement. Meanwhile, both 5aR-1 and5aR-2 can become overexpressed in the scalps of men or women who aregenetically susceptible to androgenic hair loss. As will be explained ingreater detail below with reference to FIG. 7A, the graphs show theoverexpression of 5αR-1 and 5αR-2 in women with normal hair growthversus women with female pattern hair loss (FPHL).

Both testosterone and DHT can bind to and activate androgen receptors,but DHT is more potent. Activation of androgen receptors leads to geneexpression. Since DHT is more potent, it induces higher levels of geneexpression when it activates androgen receptors, compared to the lesspotent testosterone. Overexpression of 5αR enzymes in the prostates ofaging men or in the scalps of individuals susceptible to androgenic hairloss leads to the overproduction of DHT. More DHT means an even greaterresponse in gene expression. That imbalance in gene expression can thenlead to abnormal function in the tissue, like prostate enlargement orhair loss.

Certain fatty acids found in saw palmetto extract can inhibit the 5αRenzymes in the prostate and scalp as shown in FIG. 3 and explained ingreater detail below. Fatty acids can exist in the free, unbound form,or bound to other molecules, like to glycerol forming triglyceride, forexample.

The combination of the free fatty acids plus bound fatty acids is thetotal fatty acid level.

Identification: Unlike animal oils or most vegetable oils which containfatty acids mainly in the form of triglycerides, saw palmetto oilextract is rich in free fatty acids. One method of identifying authenticsaw palmetto extracts from imitation or adulterated extracts is to testthe free fatty acid levels as a percentage of the total fatty acidlevels. High-quality saw palmetto extracts contain ≥70% free fattyacids.

Absorption: Free fatty acids are absorbed twice as well astriglycerides.

Disposition: Once absorbed into the body, free fatty acids tend tolocalize to the prostate and scalp. Why? Because cells with highexpression of androgen receptors, like those in the prostate or scalp,also express fatty acid transporters on the cell surface. Thosetransporters preferentially pull free fatty acids into the cell insteadof bound fatty acids.

Activity: Free fatty acids are the active ingredients in saw palmettoextracts. Certain free fatty acids are potent inhibitors of the 5αRenzymes, versus other forms of fatty acids which are inactive, as shownin FIG. 3 with the esterified fatty acids and fatty alcohols.

In particular, lauric, myristic, oleic, linoleic, and linolenic acidsappear to be most effective at inhibiting 5αR enzymes in the prostate,while linoleic, linolenic, and oleic acids seem most effective atinhibiting 5αR enzymes in the scalp. Therefore, increasing the overallfree fatty acid levels in an LSESr enhances the quality, the absorption,and the localization of fatty acids to the prostate and scalp.Increasing the levels of certain free fatty acids should enhance thepotency of the LSESr in specific tissues.

An LSESr that is more effective at inhibiting 5αR enzymes in theprostate should be more effective in maintaining normal urinary andprostate function in aging men than a standard extract. An LSESr that ismore effective at inhibiting 5αR enzymes in the scalp should be moreeffective in promoting healthy hair growth in individuals susceptible toandrogenic hair loss than a standard extract.

The preprocessing and milling help obtain a desired particle size andsurface area where about 10% is less than about 4 mesh and about 95%less than about 12 mesh powder and help open up the matrix to optimizethe free fatty acids as in the enhanced LSESr. Some prior art CO₂extraction parameters operate at 100-350 bar, which has not been foundadequate, especially since the process may be single step. Chemicaltransformation and alkyl hydrolysis has not been found beneficial sinceit may erode quality.

The enhanced LSESr of the current invention aids in balancing androgensignaling and controlling inflammation. The free fatty acid oleic acidhas been found especially effective for 5a-Reductase-1, which isimportant for hair health, and not as much for 5a-Reductase-2, whilelinoleic free fatty acid has been found beneficial for both, which isbeneficial for hair health treatment. Esterified fatty acids have beenfound ineffective. Linolenic acid has been found beneficial for both5α-Reductase-1 and 5α-Reductase 2, and thus effective in hair health.The preprocessing of the saw palmetto berries into the saw palmettopowder helps open up the cell structure, and the aging, such as at least15 days, and in an example, about 15 to 30 days of ageing, together withother factors, helps give the saw palmetto powder a beneficial moisturelevel, which aids during the CO₂ extraction and separation form theenhanced LSESr. There are positive benefits such as the enhanced LSESraiding in stem cell signaling, which helps influence hair growth and thehair growth cycle.

For hair health, the inventors have discovered that linoleic andlinolenic free fatty acids are beneficial and more active thanpreviously understood by those skilled in the art. It is known that DHTmay inhibit WnT signaling, which among the signaling molecules, promotesthe differentiation of hair follicle stem cells. The enhanced LSESrhelps block 5α-Reductase activity thereby reducing release of DHT thatwould otherwise inhibit WnT signaling, which plays a key role insimulating hair follicle stem cell regeneration.

An example of producing a LSESr which may be processed for an enhancedLSESr is now described. As shown in the flowchart, dried saw palmettoberries are supplied (Block 102), fed into a feed hopper (Block 104),and metallic particles and other magnetic particles or objects may bemagnetically separated from the berries (Block 106). A hammer mill maybe used to crush and mill the dried saw palmetto (Block 108). Coolingmay be maintained to prevent excessive heating during milling by theapplication of liquid nitrogen to the hammer mill as a non-limitingexample (Block 110). CO₂ extraction begins (Block 112) using carbondioxide in an example (Block 114). Spent marc (Block 116) may includesome organic residue and have some end use purposes, and thus, may beretained or discarded. CO₂ extraction includes a first fractionation(Block 118) and forming a first extract (Block 120), followed by asecond fractionation (Block 122), and forming a second extract (Block124), and a third fractionation (Block 126), and forming a third extract(Block 128), with recycling of the carbon dioxide (Block 114).

The method as described generally includes drying the berries, grindingthe dried berries, extracting the berries with the CO₂ under relativelyhigh pressure, and separating the extracted components from the CO₂.When ripe berries are harvested, they may contain about 66% water andmay be dried for several days at 130-140° F. In the resulting dry state,the berries may be stored for several years without furtherdeterioration. Dried berries may be bagged and shipped to processingfacilities where they are ground to a fineness where about 100% are lessthan about 4 mesh and about 95% less than about 12 mesh into a finepowder.

Supercritical CO₂ fluid extraction may be accomplished in an extractorvessel by contacting the ground saw palmetto berries as a fine sawpalmetto powder with a continuous flow of CO₂ at an extraction pressureof at least about 300 bar, and at a temperature lower than about 85° C.to thereby extract saw palmetto components from the CO₂. The processincludes separating the extracted saw palmetto components from the CO₂in a series of separator vessels by collecting the carbon dioxide afterextraction and decreasing the pressure in a decreasing, stepwise manner.

In an example, the extraction vessel may be loaded with the ground,dried saw palmetto berries as a saw palmetto powder, and the CO₂ may bedelivered into the vessel in a manner where the CO₂ flows through theloaded product before being discharged from the vessel. As thehigh-pressure CO₂ flows through the product, it behaves as a lipophilicsolvent and extracts those saw palmetto lipidosterolic components thatare soluble in the fluid. Different extraction pressures may be used,but it has been found that an extraction pressure of about 500-550 baris advantageous. A first separation pressure may be about 250-300 bar,and may be about 160 bar to about 350 bar. Extraction may be conductedat a temperature from about 45° C. or 50° C. to about 90° C., andpreferably under a substantially continuous flow of carbon dioxide. Inan example, the first fractionation (Block 118 in FIG. 1 ) may be about35° C. to about 85° C.

The method may include the second fractionation (Block 122 in FIG. 1 )and associated separation having a separation pressure lower than thefirst separation pressure to separate a second fraction of dissolvedcomponents and compounds from the carbon dioxide. The second separationmay include the fractionation as a separation pressure of about 50 toabout 200 bar, and in another example, about 120 to about 150 bar. Thetemperature may range from about 20° C. to about 80° C.

Additionally, in accordance with another embodiment, the method mayinclude a third fractionation (Block 126 in FIG. 1 ) and associatedseparation having a separation pressure lower than the second separationpressure to separate a third fraction of extracted compounds from thecarbon dioxide. Sequentially, the CO₂ flows into a third separationvessel and the third fraction is collected. The third separation mayinclude a separation pressure of about 30 to about 80 bar, and inanother example, about 30 to about 60 bar, wherein the CO₂ is in thegaseous state. Following the last separation, the CO₂ is returned tostorage for further use.

Plural separations may be conducted sequentially and in a substantiallycontinuous flow. Each subsequent separation may have a lowerpredetermined separation pressure and operate as sequential separationsreferred to as cascading separations. Sequential separations separatethe extracted saw palmetto compounds from the carbon dioxide into aplurality of fractions. Passing the substantially continuous flow ofcarbon dioxide after extraction through the different separations willseparate the extract into several fractions.

In a preferred technique, each individual fraction of the resultingplurality of fractions may have a different fraction of extracted sawpalmetto components from the other individual fractions. For example,the first fraction may include a major fraction of saw palmetto steroland triacylglyceride compounds, the second fraction may include afraction of saw palmetto components similar to a whole extract, and athird fraction may include a major fraction of saw palmettounesterified, free fatty acids. This is contrasted where acylglyceridecompounds include esters derived from glycerol and 1 - 3 fatty acids.

An advantageous benefit of the method as described is the ability toblend the resulting extraction fractions at predetermined amounts toprepare a dietary supplement composition that includes desiredproportions of different saw palmetto components, including sterols,unesterified free fatty acids and fatty alcohols. This method allows notonly the preparation of custom blends to meet specific consumer demands,but also the preparation of a dietary supplement composition that hasbeen standardized for the final end use of the product.

The resulting dietary supplement composition may be formulated for oraluse and nutritional supplementation. The composition may include apharmaceutically acceptable carrier and may be orally administered as agel capsule containing a unit dose. In an example, the daily dose maycontain about 160 mg b.i.d. to about 320 mg, or at least about 200 mg ofsaw palmetto lipids as an enhanced lipidosterolic extract of Serenoarepens, (LSERs), and in another example, the unit dose may contain about320 mg of saw palmetto lipids as enhanced effectiveness from LSERs for asingle daily unit consumption.

A method for use may include ingesting the dietary supplementcomposition having at least about 85% saw palmetto fatty acids as theenhanced LSESr, and preferably greater than about 90% of these fattyacids present as free, unesterified fatty acids. The method for use mayinclude ingesting the dietary supplement composition that hasessentially no solvent residue. Ingesting may be accomplished byswallowing a capsule unit dose that includes a pharmaceuticallyacceptable carrier. A suggested daily dose may be about 320 mg of sawpalmetto lipids as a LSESr, or about minimum 200 mg per dose of enhancedLSESr, preferably having a relatively high amount of unesterified fattyacids greater than about 72%.

An example of the production parameters and resulting extractioncomponents from the extraction and isolation of the saw palmetto lipidsbased upon the process described relative to the flowchart in FIG. 1 isshown in Table 1. This table summarizes an example of the chemicalcomposition of lipidosterolic extract of Serenoa repens (LSESr) createdby the sequential separations as described above.

Table 1 Parameter 1st Separation Fraction 2nd Separation Fraction 3^(rd)Separation Fraction All Fractions Combined Separation Pressure (bar) 290140 55 Separation 60 70 25 Temperature (°C) Fraction Mass (kg) 11 701 74786 Acid Value (mg KOH/g) 83 184 209 185

Fatty Acid (% w/w) Caproic 0.6 1.2 2.1 1.3 Caprylic 0.9 2.1 3.3 2.2Capric 1.1 2.6 3.4 2.7 Lauric 12.5 26.1 30.9 26.4 Myristic 5.7 10.6 11.210.6 Palmitic 5.7 8.8 8.2 8.7 Palmitoleic 0.1 0.2 0.2 0.2 Stearic 1.21.7 1.4 1.7 Oleic 22.7 32.8 28.1 32.2 Linoleic 3.0 4.6 3.7 4.5 Linolenic0.7 0.5 0.4 0.5 Total Fatty Acids 54 91 93 91 Percent as Free FattyAcids 62 85 90 85

Sterols (% w/w) Stigmastanol 0.029 0.010 0.006 0.010 Campesterol 0.1220.055 0.036 0.054 Beta-Sitosterol 0.398 0.166 0.107 0.164 Stigmastanol0.054 0.025 0.017 0.025 Total Sterols 0.603 0.256 0.166 0.252

Long-Chain Alcohols (% w/w) 0.011 0.003 0.002 0.003 TetracosanolHexacosanol 0.036 0.017 0.010 0.017 Octacosanol 0.245 0.162 0.087 0.156Triacontanol 0.048 0.031 0.016 0.030 Total Alcohols 0.340 0.213 0.1150.206

During the supercritical carbon dioxide fluid extraction, it may bepossible to use entrainers as cosolvents, although this is not necessaryto obtain the desired LSESr. It is also possible to modify the phasebehavior of supercritical solvents and enhance solubility of thecomponents in the carbon dioxide-rich phase. A cosolvent may alter thepolarity, viscosity and density of any gas phases. It may be possible touse cosolvents such as alcohols and hydrocarbons (straight-chained andbranched) that may have a higher molecular weight. It may be possible touse ethoxylated compounds.

The enhanced lipidosterolic extract of Serenoa repens (LSESr) asmanufactured and described may be formulated for prostate health or hairhealth, including addressing androgenic hair loss. The enhanced LSESrhas been shown to inhibit 5α-Reductase 1 and 2. The enhanced LSESr mayspecifically target prostate tissue, reduce urinary frequency, andsupport, maintain and promote healthy urinary and prostate function. Theenhanced LSESr may support optimal urinary flow and control in men andinhibit 5α-reductase without sexual side effects.

This enhanced lipidosterolic extract of Serenoa repens may be formulatedfor hair health treatment and regrowth because the enhanced LSESr hasbeen found to target 5α-Reductase 1 and 5α-Reductase 2, which are foundin hair follicles and affect hair regrowth and thickness.

The enhanced LSESr has a high percentage of free fatty acids notcommonly found in nature. The enhanced LSESr has markedly differentcharacteristics with a marked change in function and activity to treatsymptoms of prostate disorder and promote urinary and prostate function,or for hair loss in a patient to promote prostate health and hairregrowth. The different free fatty acids as fractions alone or incombination provides a marked change in function and activity comparedto what is normally found in nature. These benefits are accomplished viathe plurality of separations and resulting manufacture of severalfractions.

The enhanced lipidosterolic extract of Serenoa repens (LSESr) asmanufactured and described may be encapsulated in a gelatin, carrageenanor starch based soft gel for oral consumption. The enhanced LSESr may becombined with dry excipients including, but not limited to one or moreof silicon dioxide, calcium silicate, calcium phosphate, magnesiumoxide, magnesium carbonate, calcium carbonate, rice fiber, andmaltodextrin to create a free-flowing powder form of the extract. Theenhanced LSESr may also be combined with excipients and spray dried intoa free-flowing powder. The enhanced LSESr may also be combined withwater and emulsified using excipients to create a stable emulsion, whichmay be dried such as by spray drying, belt drying, vacuum drying, orfreeze drying to create a powder form.

The different carriers and formulations may also impart markedlydifferent characteristics for the enhanced LSESr to stabilize thecomposition and impart functions that are significantly more in functionthan found in nature, and help in bioavailability of the compositionthat includes the enhanced LSESr when orally digested. It is possible touse a carrier for contacting the skin to help reduce acne.

Other additives may be included with the enhanced LSESr and may includepumpkin seed, Vitamin D, zinc, and rosemary oil, alone or in differentcombinations. The enhanced LSESr has been found to be stable and notrequire added antioxidants and has extended shelf life. Other additivesmay be included, such as Valensa’s O2B® Peroxidation Blockerstabilization technology and other components, including astaxanthin,phenolic additives, and natural and synthetic tocopherols andtocotrienols, carnosic acid or carnosol and/or astaxanthin, but evenwithout these additives, the enhanced LSESr has an extended shelf lifeas noted before.

Other additives with the enhanced LSESr may include a mixture ofselected lipophilic and hydrophilic components. Lipophilic additives maybe used either alone or in combination with at least one of: a) phenolicadditives including at least one of sage, oregano, and rosemary; b)tocopherol(s); c) tocotrienol(s); d) carotenoids including at least oneof astaxanthin, lutein, and zeaxanthin; e) ascorbyl acetate; f) ascorbylpalmitate; g) Butylated hydroxytoluene (BHT); h) Butylatedhydroxyanisole (BHA); or i) Tertiary Butyl hydroquinone (TBHQ). Ahydrophilic additive may include a sequestrant and may includehydrophilic phenolic additives including at least one of grape seedextract, tea extracts, ascorbic acid, citric acid, tartaric acid, andmalic acid.

The process for manufacturing the enhanced LSESr as described above,such as with reference to the flowchart of FIG. 1 , may be modified,such as shown in the sequence described at FIG. 55 , to break outdifferent fatty acids and free fatty acids that are important to promoteurinary and prostate functions in men with an enlarged prostate, andenhance and maintain hair health and growth and reduce androgenic lossin a susceptible individuals. It is possible that humans or animals mayconsume the enhanced LSESr to be treated.

An example of the enhanced LSESr is shown in the table of FIG. 4A andExperiments 5 and 6 as Lot Nos. 211022 and 211105 and a secondaryenhanced LSESr as Lot No. 210908, but having a lower ratio of fouractive free fatty acids. For example, some fatty acids play a moreimportant role as more active free fatty acids in enzyme inhibition,such as 5aR-1 and 5aR-2 enzymes. The inventors have discovered thatthere are four free fatty acids that have greater influence and activityin the enhanced lipidosterolic extract of Serenoa repens (LSESr), andrecognize these more important and active free fatty acids as lauric,myristic, oleic, and linoleic free fatty acids, and a fifth as linolenicfree fatty acid that may be enhanced even more for hair health. Thesefive key active free fatty acids aid in prostate health, hair growth,and skin, e.g., acne prevention. Of these five free fatty acids, someskilled in the art consider linolenic to be of reduced importance, butthe inventors have discovered the beneficial aspects of linolenic acidas a free fatty acid to aid in inhibiting both 5α-Reductase Type 1 andType 2 and help in hair growth, especially in combination with the freefatty acid linoleic acid.

FIGS. 2 and 3 are tables that respectively show the total 5α-reductase50% inhibition (IC₅₀ µg/ml) of the 5α-R1 and 5α-R2 enzymes to converttestosterone to DHT by various constituents found in saw palmettoextract (FIG. 2 ), and the magnitude of the effect by the variousconstituents on a comparative basis (FIG. 3 ). The different free fattyacids of medium and long saturated chains and long unsaturated chainsare shown, together with esterified fatty acids, oxidized fatty acids,fatty alcohols, phytosterols, and other components. The magnitude of theactivity is designated by “none,” “moderate” or “very active” (FIG. 3 ).The various constituents shown in the table of FIG. 2 are also shown andlisted as free fatty acid molecules with: a) medium, saturated chains,b) long, saturated chains, and c) long unsaturated chains. There areesterified fatty acids, oxidized fatty acids, and fatty alcohols. Thesetables support other evidence that the five active free fatty acids forprostate and hair health are lauric, myristic, oleic, linoleic andlinolenic free fatty acids.

The major contributors of active fatty acids unique to the enhancedLSESr are largely free fatty acids. The free fatty acids in the enhancedLSESr are uniquely intensified compared to other forms of fatty acidsfound in other sources. Vegetable oils contain fatty acids primarily inthe triglyceride form, whereas LSESr contain primarily free fatty acids.In addition, LSESr contain high levels of lauric acid. Most vegetableoils, with the exception of coconut oil, typically do not contain lauricacid.

It is known that free fatty acids, such as derived from fish oil, arebetter absorbed and more bioavailable than correspondingtriacylglycerides or ethyl esters that may be found in fish and similaroils. For example, absorption may occur at greater than 95% for the freefatty acids, versus 57% to 68% for the triacylglycerides, or versus 20%to 21% for the ethyl esters, respectively. There are some saw palmettoextracts that have high levels of free fatty acids, while other sawpalmetto extracts have lower levels of free fatty acids. For example,depending on the type of extraction technique applied to saw palmettoberries or ground saw palmetto, the levels of free fatty acids in sawpalmetto extracts may vary from 40% to 80%, but typically never beyond80% even with supercritical CO₂ extraction. Saw palmetto extracts can beexpensive to produce, and for that reason, there are known vegetableoils that are commonly used to adulterate or imitate saw palmettoextracts, but these oils contain high levels of triglycerides. Those sawpalmetto extracts having higher free fatty acid concentrations arebetter absorbed and made more bioavailable than saw palmetto extractshaving lower levels of free fatty acids and vegetable oil comparators.Once absorbed in the body, some free fatty acids specifically target theprostate, or the scalp to aid in hair health and hair growth.

There are some factors making free fatty acids undesirable in vegetableoils, even though they are made more bioavailable when ingested, forexample. Free fatty acids are highly reactive and may create stabilityissues, and vegetable oils may degrade quickly. For example, heat andfrying may degrade vegetable oils, and thus, quality is monitored bytesting peroxide value as a measure of oxidative degradation.

This issue of oxidative degradation is not as challenging with sawpalmetto extracts because the oil extracted from the saw palmetto isacidic, unlike vegetable oils, which has a more neutral pH. This benefithas been realized by the inventors with the enhanced LSESr of theinvention. For example, the enhanced LSESr has a four-year shelf lifethat is a technical achievement for an oil that is high in free fattyacids, and especially with the higher ratio of free fatty acids to totalfatty acids. This higher ratio of free fatty acids to total fatty acidsand especially the contribution of four free fatty acids of lauric,myristic, oleic and linoleic was accomplished by the uniquepreprocessing of saw palmetto berries in combination with the uniquesupercritical CO₂ processing parameters. It is feasible to achieve highfree fatty acid oil concentrations with an acidic pH, and this benefitof higher concentration is aided because the saw palmetto berries act asa natural oil protection, while excluding oxygen, without having toartificially stabilize the free fatty acids. For example, in an exampleof the enhanced LSESr, the peroxide value (PV) specification is below5.0 meq/kg, and is below 1.0, so oxidative damage is controlled as well.

Based on a lipid chemistry perspective, the higher the free fatty acidsare driven, the more likely there will be stability issues encountered.However, in accordance with a non-limiting example of the invention, thepreprocessing of saw palmetto berries as described with the improvedsupercritical CO₂ extraction and enhanced processing parameters, thestability issue because a more minor issue because the supercritical CO₂extraction process is a closed system in the absence of oxygen and usedwithout organic solvents, such as hexane. This may be distinguished witha known commercial extraction with the organic solvent hexane as used tomanufacture the commercially available European extract known asPermixon® with its attendant technical drawbacks as explained below.

Enhanced processing parameters for supercritical carbon dioxideextraction is employed when manufacturing the enhanced LSESr of thecurrent invention rather than hexane, and this permits the process toachieve a stable extract which is “naturally/non-organic solvent”stable. Together with unique preprocessing of the saw palmetto berries,the enhanced LSESr is produced. Thus, any composition that includes theenhanced LSESr achieves a balanced fatty acid profile that is rich infree fatty acids, room temperature stable and stable for years.

Referring now to the tables shown in FIGS. 4A and 4B, and the graphs andbar charts of FIGS. 5 and 6 , and Inhibition Experiment 1 (FIG. 5 ) andInhibition Experiment 2 (FIG. 6 ), the inhibition of 5α-Reductase 2 isshown, which is the primary 5α-Reductase isoenzyme active in prostatetissue. The tables of FIGS. 4A and 4B show the results for a lipidprofile comparison of a commercially available hexane extract Permixon®,and six further experiments of supercritical CO₂ extraction with varyingparameters. Experiments 1, 5 and 6 had similar preprocessing of berries,but Experiment 1 used a low-pressure CO₂ extraction process typicallyused for extraction of hops. This lower pressure extraction extractedfewer mono- and diglycerides and unsaponifiable matter than the uniquehigh pressure CO₂ extraction process such as with Experiments 5 and 6.

The conclusions for the biological assay are shown in FIG. 4B, whichindicate that Experiments 5 and 6 as Lot Nos. 211022 and 211105 producethe enhanced LSESr and has the better profile to achieve the desirablerange and percentage of total fatty acids and the better proportion offree fatty acids with their proper balance, ratio and enrichment ofselected free fatty acids as lauric, myristic, oleic and linoleic freefatty acids. Their percentage was greater than 82.0%. Experiment 4 is asecondary enhanced LSESr with the lower ratio of the four active freefatty acids, but was still found to be effective, but not as much as theLSESr from Experiments 5 and 6.

If the concentration of free fatty acids is too high, it may be moredifficult to obtain enough enrichment in the four important fatty acidsthat drive biological activity, i.e., the lauric, myristic, oleic, andlinoleic free fatty acids. For that reason, one goal was to achieve therange of about 82.0% to about 84.0% for these four bioactive free fattyacids. This can range from about 82.0% to as high as 86.0%, butpreferably about 82.0% to about 84.0%. The better optimization comesfrom supercritical CO₂ extraction control and balance, such as describedwith reference to the process shown and described relative for theprocess shown in FIG. 55 , that can be achieved with the rightenrichment. In an example, the enhanced LSESr may have a balance ofabout 90.0%, and in a specific example, about 88.9% to about 90.2% oftotal fatty acids to about 72.0%, and in a specific example, about 72.4%to about 72.6% of free fatty acids. The enhanced LSESr had a ratio offree fatty acids to total fatty acids of about 80.0% to about 82.0%.

Experiment 1 as U4868 was a lower pressure and temperature extractionprocess such as typically used for extraction of hops products, e.g.,around 150 bar and 40° C. The Experiment 1 low pressure process hadquality issues by not removing all components, such as the mono anddiglycerides and unsaponifiable matter. Although it may develop a ratioof free fatty acids to total fatty acids greater than the commerciallyavailable Permixon® and even some fatty acid ratios similar to theenhanced LSESr of Experiments 5 and 6, the lower pressure processproduced an extract product differing from the invention in Experiments5 and 6. The greater than 80% ratio of free fatty acids to total fattyacids and higher percentage of the four active free fatty acids wasachieved for the enhanced LSESr shown by Experiments 5 and 6 as Lot Nos.211022 and 211105, and had excellent shelf stability and as much as 4years stability without added antioxidants and stabilizers. This wasalso achieved by the enhanced berry preprocessing with aging of thecryogenically milled saw palmetto berries and the CO₂ extractionparameters as later described.

Experiment 3 had similar CO₂ extraction processing parameters withslight differences, but no enhanced preprocessing of berries withspecific cryogenic milling and aging of saw palmetto powder as withExperiments 5 and 6. Thus from those test results, it was evident thatbiological activity for the LSESr of Experiments 5 and 6 was not drivenby simple factors of total fatty acids, free fatty acids, or the levelof the important fatty acids as free fatty acids. The better profile ofthe enhanced LSESr was driven by the more desirable and inventivepre-extraction handling parameters described below and the inventivesupercritical CO₂ extraction and separation parameters on the ripeberries, such as described relative to the flowchart of FIG. 55 , toachieve: 1) a higher ratio of delivered free fatty acids to total fattyacids, which also aids in inflammation reduction; and 2) at the sametime, a higher enrichment of the four important fatty acids as freefatty acids to total free fatty acids, which value is higher than theother compared products, including the commercially availablePermixon®.The product obtained from Experiment 1 as the extractedproduct from the low-pressure process (U4868) has a lower ratio of thefour beneficial free fatty acids compared to the enhanced LSESr ofgreater than 82.0%.

Comparing the percentages across the different lots, the total fattyacids is less important than the ratio of total fatty acids to freefatty acids. What is also important is the optimization of thecontribution of the four bioactive fatty acids (lauric, myristic, oleic,and linoleic) as a percentage of total free fatty acids. Thesebeneficial values in the enhanced LSESr are driven by the inventivepreprocessing parameters for the saw palmetto berry and supercriticalCO₂ extraction parameters. A fifth bioactive free fatty acid, linolenic,also contributes especially for hair health and operates to help reduceinflammation and aid signaling. It is possible to achieve a similarpercentage of free fatty acids to total fatty acids from proportionallylower total fatty acids or higher free fatty acids. For example,Experiment 1 as the low pressure process (U4868) delivered more of thesetotal free fatty acids at 87.0% and the contribution of free fatty acidsto total fatty acids at a higher 94%, but less than 82.0% for the activefour free fatty acids as in Experiments 5 and 6.

The aging, milling, and particle size deliver the optimum ratio of 4(four) critical free fatty acids to total fatty acids via thesupercritical CO₂ extraction. The invention and optimizedaging/moisture, particle size and extraction parameters drive the valueof the four active free fatty acids to total free fatty acids, notdependent on achieving highest total fatty acids or free fatty acids.The preconditioning of the berries allows the extraction process toachieve the inherently beneficial profile.

The 5α-Reductase Type 2 50% (IC₅₀) inhibition data are evident in thegraphs and bar charts of FIGS. 5 and 6 for the two 5α-Reductase 2Inhibition experiments, which compared Experiment 2 (U4602) as a moreconventional CO₂ extract for an LSESr to inhibit 5α-Reductase 2 versuscommercially available Permixon®, and the saw palmetto extract asExperiment 1 (U4868) corresponding to the low pressure. Data are shownin the graphs and bar charts of FIGS. 5 and 6 where data are reported asthe effectiveness to inhibit enzyme activity (IC₅o). InhibitionExperiment 2 (FIG. 6 ) examined the possible impact of the commerciallyavailable Permixon® product formulated ona pegylated (PEG) base ormatrix with the conventional CO₂ extracted product in Experiment 2 thathad been also formulated with polyethylene glycol(U4602-PEG) .

In Inhibition Experiment 1 (FIG. 5 ), it should be understood that theuse of the extract from Experiment 2 (4602-1) was a high-pressuresupercritical CO₂ extract and the extract derived from a modifiedsupercritical CO₂ extraction in Experiment 1 was produced at a lowerextractor temperature and pressure, but used berries that had beenpreprocessed in a similar manner as used to produce the enhanced LSESrof the current invention and described below. Even though there was alower ratio of the four active free fatty acids, it did have a highcontribution of free fatty acids to total fatty acids of about 94.0%,but that did not make up for effectiveness. The Permixon® product was ahexane saw palmetto extract sold as a commercial product on a pegylatedmatrix as indicated. At concentrations of about 1.0 to 70 ug/ml, it isevident that the supercritical CO₂ extracts have greater percentage ofinhibition than the commercially available hexane extract preparation,Permixon®, but at above 70 µg/ml, the percentage inhibition is about thesame for all three saw palmetto extracts.

Inhibition Experiment 2 shown in FIG. 6 determined the possible impactof Permixon® product matrix (PEG) on an in vitro assay and the pegylatedand conventional ultra-high purity supercritical CO₂ extract having thePEG matrix (U4602-PEG) was compared to the commercially availablePermixon® and the conventional unpegylated product (U4602) for theirability to inhibit the 5α-Reductase 2 enzyme. The differences ininhibition were not as pronounced as compared to the samples of FIG. 5 ,indicating that the PEG matrix has an effect.

Lipid extracts manufactured using conventional techniques such as hexaneextraction (Permixon®) or ethanol extraction (SABALSELECT®) from ripesaw palmetto berries typically contain over about ca. 70% free fattyacids. The experiments and studies show that ripe saw palmetto berriesthat have been preprocessed using techniques of milling and grinding asdeveloped by the inventors and coupled with selected CO₂ extraction andseparation pressures and temperatures as developed by the inventors maybe employed to optimize the free fatty acids, which has been found to bea key to effectiveness, such as accomplished with the enhanced LSESr ofExperiments 5 and 6 as Lot Nos. 211022 and 211105. Both the total andfree fatty acids are important, but the higher level of free fatty acidsand especially the four bioactive free fatty acids of lauric, myristic,oleic and linoleic are more biologically more relevant. Experiments havealso shown that only certain types of fatty acids inhibit 5α-Reductaseisoenzymes Type 1 and Type 2, such as the four primary more bioactivefree fatty acids of lauric, myristic, oleic, and linoleic acids, andwith a fifth as linolenic free fatty acid in combination with linoleicacid, and each of the free fatty acids having a different effect whetherit is 5α-Reductase Type 1 or Type 2 enzyme.

Linolenic acid has been found advantageous for hair health incombination with linoleic acid as free fatty acids and inhibiting5α-Reductase Type 1 enzyme and as an aid in signaling. These free fattyacids are believed to act synergistically in inhibiting 5α-Reductaseboth Type 1 and Type 2. The overall lipid profile of the enhanced LSESrconfirms authenticity. By establishing pre-extraction processingparameters of ripe berries, together with the production parameters forsupercritical CO₂ extraction, it is possible to ensure that the desiredfree fatty acid concentrations and ratios, especially with the fourbioactive free fatty acids, are met to form the enhanced LSESr. Thus, aquality product may be produced such as the enhanced LSESr. Biologicalassays will not be required for every production lot since the repeateduse of the preprocessing parameters of the saw palmetto berries and thespecific supercritical CO₂ extraction parameters will repeatedly producethe enhanced LSESr having a ratio of free fatty acids to total fattyacids that is greater than about 80.0% and an enrichment of lauric,myristic, oleic and linoleic free fatty acids that is greater than about82.0%.

In an example, this enhanced LSESr may have a balance of about 90.0% oftotal fatty acids to about 72.0% of free fatty acids, and an enrichmentof the four active free fatty acids of about 82.0% to about 84.0%. Ithas been found the ratio of free fatty acids to total fatty acids ofabout 80.0% to about 82.0% is effective with about 82.0% to about 84.0%of the four bioactive free fatty acids.

The fatty acids in the LSESr target the prostate. The 5α-Reductaseenzymes are found in several different tissues in the body. However, thefree fatty acids in the enhanced LSESr target the prostate cells, andunlike most cells in the body, the prostate cells preferentially uptakethese free fatty acids instead of glucose. The androgen receptors upregulate fatty acid transporters on the surface of prostate cells.

It has been found that there is a difference in function betweenFinasteride as a 5α-Reductase inhibitor and the enhanced LSESr bioactivefree fatty acids. Finasteride permanently locks the enzyme closed, andcompetitively blocks the binding sites. The enhanced LSESr bioactivefree fatty acids, on the other hand, temporarily change the make-up ofthe nuclear membrane, which disrupts the stability of the enzyme, andnon-competitively blocks the binding sites. This is one reason why thetype, quantity and ratio of the free fatty acids relative to fatty acidscan be important and slight variations, such as found with the enhancedLSESr of the invention as compared to other CO₂ extracted products orPermixon® or other hexane extracted saw palmetto extracts can have sucha positive difference in function.

By specifically targeting prostate cells, the enhanced LSESr has noeffect on circulating DHT (Dihydrotestosterone) levels and has low sideeffects, including low sexual side effects. The free fatty acids in theenhanced LSESr change the lipid composition of the nuclear membrane,which disrupts 5α-Reductase activity. The impact on the 5α-Reductaseenzymes is reversible and non-competitive, while the biological functionof the 5α-Reductase enzyme is conserved. These effects are not seen withvegetable oils or saw palmetto imitators because these oils have thewrong forms of fatty acids and free fatty acids, the wrong ratios, andthe wrong lipid profile. Powdered saw palmetto products are very common,but they do not have enough lipid content, and often have an inferiorlevel and type of fatty acids.

The free fatty acids are inserted into the prostate cell nuclearmembrane and impact functionality of that cell nuclear membrane, disrupt5α-Reductase enzyme activity, lead to no further or limited enlargementof the prostate, lead to less inflammation with a resultant improvementin urinary flow and control, and no delayed progression of thecondition. Small changes in amounts, ratios and specific free fattyacids make large differences as the inventors have determined, such aswith the four bioactive free fatty acids, enabling peak results for theenhanced LSESr. The free fatty acids of the enhanced LSESr operate asactive ingredients for clinically proven anti-inflammatory andanti-proliferative effects, which are likely due to reduced inflammatoryenzyme activity such as 5-lipoxgenase, COX-2, Phospholipase A₂, andsimilar enzymes via disruption of the cell nuclear membrane. Theinhibition of the NF-kB activation reduces inflammatory gene expression.

These effects and functions can be explained when comparing a balancedimmune system and an unbalanced immune system. For example, a balancedimmune system responds to triggers such as an infection or injury andactivates pro-inflammatory enzymes and pro-inflammatory mediators todeal with the threat. A balanced immune system also resolvesinflammation after the threat has been neutralized, which promoteshealing of tissues by increasing production of growth factors. Anunbalanced immune system, on the other hand, does not resolveinflammation, even after the threat is no longer present. An unbalancedimmune system may have an uncontrolled activation of pro-inflammatorymediators, which leads to tissue damage and dysregulates production ofgrowth factors, resulting in abnormal cell proliferation and tissueremodeling. Hypoxemia may occur from rampant cell growth that leads toangiogenesis and production of more growth factors.

The enhanced LSESr of the current invention disrupts the activity ofpro-inflammatory enzymes and the expression of inflammatory genes.Similar to 5α-Reductase, the enhanced LSESr changes the composition ofthe cell nuclear membrane, which disrupts the activity ofpro-inflammatory enzymes. By blocking activation of NF-kB, the enhancedLSESr reduces the expression of numerous inflammatory mediators such aspro-inflammatory enzymes, adhesion molecules, cytokines, chemokines,growth factors, receptors, and transcription factors.

It is understood that the fatty acid transporters on prostate cellspreferentially take up the free fatty acids instead of glucose, unlikemost cells in the body. Thus, the free fatty acids of the enhanced LSESrinhibit the 5α-Reductase and are anti-inflammatory. The enhanced LSESrof the current invention is rich in free fatty acids and their specificcombination and ratio. An example is the enhanced LSESr of Experiments 5and 6 (FIG. 4A), which is able to support prostate and urinary health byrestoring balance in the androgen signaling such as caused by the staticobstruction and DHT mediated prostate enlargement, and addressinflammatory pathways in the prostate. The enhanced LSESr of the currentinvention has a sexual side effect level similar to baseline or placebosused in other trials. Concerning possible drug-drug interactions, theenhanced LSESr of the current invention does not appear to affect themajority of the cytochrome P450 isoenzymes and does not interfere withPSA antigen testing. The enhanced LSESr does not reduce the secretion ofPSA, even in long-term studies.

Reference is made again to FIGS. 4A, 4B, 5 and 6 and the discussion ofInhibition Experiments 1 and 2. In Inhibition Experiment 1 (FIG. 5 ),two CO₂ extraction procedures were employed, i.e., one using the lowerpressure CO₂ extraction (U4868) as Experiment 1 in FIG. 4A and aconventional CO₂ extraction of Experiment 2 in FIG. 4A (U4602) andcompared to Permixon®, and studied for the inhibition of 5α-ReductaseType 2 enzyme as primarily reflected in prostate tissue. It is believedthat the difference in inhibition rates may be due to the presence ofthe polyethylene glycol (PEG) in the Permixon® formulation, which maybind to the free fatty acids and subsequently reduce the amount that canbe extracted in test samples.

Inhibition Experiment 2 (FIG. 6 ) further showed the differences betweenconventional supercritical CO₂ saw palmetto extracts complexed with PEGversus that saw palmetto without PEG where the pegylated sample(U4602_PEG) was generated. The pegylated sample had an IC₅₀ of 7.47+/-0.07 micrograms per milliliter (µg/ml), similar to the IC₅₀ of 7.72+/-0.05 µg/ml for the commercially available hexane extracted Permixon®,where the p-value was not significant. The unpegylated sample (U4602-1)had an IC₅₀ of 4.54 +/-0.23 µg/ml with a p-value less than 0.0001compared to each. These results show the beneficial aspects of asupercritical CO₂ extract over conventional hexane extraction, such asthe commercially available Permixon.

It should be understood that the enhanced LSESr is also beneficial forhair treatment of adrogenic hair loss and reduces inflammation and aidsin stem cell signaling. Hair growth and loss is a natural occurrencewith the growth phase referred to as Anagen where the follicles areanchored at the base. This stage occurs for about 2-6 years duration andabout 90% to about 95% of the follicles are at this stage of the hairgrowth cycle. The transition phase (Catagen) is about 2-3 weeks and lessthan 1% of hair follicles are at that stage. In this Catagen stage, thehair follicles slowly detach from nourishing blood supply and stopgrowing. The resting phase known as Telogen is about 2-3 months andabout 5% to 10% of follicles are in this stage, where there is nonourishment and the hair dies and falls out. The early Anagen phasefollows where the hair follicle re-establishes and hair regrowth begins.The duration of the growth phase imparts hair length and can be 2-6years in duration. Blood flow provides nourishment that enables growth.

When the hair growth cycle is impacted, the result is hair thinning andresultant shorter and finer hair follicles over time, such as occurswith miniaturization and alopecia. Thus, any hair loss remedy mustbalance the cycle of growth, loss, and the resting phase before growthbegins again. Humans are typically born with all hair follicles, e.g.,about 100,000 on the scalp. More hair follicles are not produced, buthair growth and hair health can be supported.

Androgens are considered the main regulators of the hair follicle.Normal hair growth cycle takes place in the presence of androgens.However, androgens may inhibit hair follicle growth in geneticallysusceptible individuals, leading to small, thin hair and hair loss, alsoknown as Androgenic Alopecia (AGA). Usually AGA is a hereditary thinningof the hair induced by androgens and may begin between the ages of 12and 40 in both sexes, but it is less severe in women due to the lowerlevels of androgens.

There is an interplay between androgens and the androgen receptor andthe hair follicle. Androgens from the blood, such as circulatingtestosterone, may enter the hair follicle via the dermal papilla’s bloodsupply and may be converted by 5α-Reductase to the more potent androgenDHT, which may bind to the androgen receptors in the dermal papillacells, causing changes in their production of regulatory paracrinefactors. This directly alters the activity of dermal papilla cells.Keratinocytes and melanocytes may also be affected. The complex thenbinds to elements in the DNA, altering the expression of specificandrogen-dependent genes.

If both testosterone and DHT are present in similar quantities, theandrogen receptor will bind 5α-dihydrotestosterone (DHT) rather thantestosterone. Once the 5α-DHT complex is formed, the receptor complexundergoes conformational change. Along with co-activating proteins, the5a-DHT complex binds to elements in the DNA, altering expression ofspecific androgen-dependent genes. The effect is the hormone-receptorcomplex-activated genes change large terminal follicles to miniaturizedfollicles. Each successive hair cycle undergoes a shorter growth phaseand the follicles become smaller, producing shorter, finer hair thatpoorly covers the scalp.

Signaling is indicative that the hair follicle stem cells move from theresting phase and start the growth phase. DHT interferes with thesignaling that begins the hair growth cycle and can activate expressionof an important gene (DKK-1), a major hair loss factor secreted fromdermal papilla (DP) cells that may induce and accelerate AGA. DHTup-regulates Catagen inducers, and the dermal papilla cells produceDKK-1 in response to DHT in the AGA scalp.

DKK-1 is a Catagen inducer and suppresses dermal papilla cells andkeratinocyte proliferation and slows the differentiation of hairfollicle stem cells. DHT also up-regulates the secretion of IL-6 andT6F-β1 by dermal papilla cells. This suppresses keratinocyteproliferation and inhibits hair shaft elongation. Scalp DKK-1 is muchhigher in individuals with AGA than evident with others serving ascontrols. Men have more 5α-Reductase enzymes and androgen receptors inthe scalp than women, which leads to higher levels of DHT-AR complex andhigher DKK-1 levels in scalps. For that reason, androgenic hair loss inmen is more severe than for women.

The 5α-Reductase enzymes Type 1 and Type 2 are located within hairfollicles, where they influence hair growth and hair miniaturization.The 5α-Reductase Type 1 enzyme is almost everywhere, while the5α-Reductase Type 2 is very localized. 5α-Reductase 1 and 2 are presentin higher levels in women with female pattern hair loss and the5α-Reductase inhibitor therapy has been shown to be effective in menwith androgenic hair loss. The hair structure includes differentcomponents. The inner root sheath anchors and protects the hair shaftand the outer root sheath surrounds the entire hair follicle and is asource of stem cells. The matrix cells generate hair and the papillaallow for vasculature for blood supply. The more localized 5α-ReductaseType 2 enzyme is not typically located in the outer root sheath andpapilla. For this reason, the enhanced LSESr may affect 5α-ReductaseType 1 enzyme, and having enhanced linolenic and linoleic free fattyacids, may be better suited for hair health.

Saw palmetto extract of about 320 milligrams per day versus finasterideof 1 milligram per day had been studied in 100 men with mild to moderateandrogenic alopecia to assess changing hair density over 24 months. Bothtreatments increased hair growth but finasteride was found moreeffective. The saw palmetto extract stabilized hair loss in 52% of men,and 38% of men treated with the saw palmetto extract had increased hairgrowth, and 68% of men treated with finasteride noted an improvement.

However, as is well known, finasteride comes with side effects not seenwith saw palmetto extracts. Inhibiting the 5α-Reductase Type 1 enzymehelps limit the production of DHT and supports hair health. The graphsin FIG. 7A show a comparison between a control and female pattern hairloss (FPHL) and how the number of molecules of 5α-Reductase Type 1enzyme increase with those having FHPL compared to a normal control asthe left-hand graph, and the right-hand graph showing that 5α-ReductaseType 2 enzymes are also overexpressed in women with FPHL.

Reference is now made to FIGS. 7B and 7C showing the use of asupercritical CO₂ extracted LSESr and Permixon®. The inhibition of the5α-Reductase Type 1 enzyme displayed in the graph and bar charts of FIG.7B show the mean percent inhibition relative to concentration (pg/ml)and mean IC₅₀ (µg/ml) in the bar chart on the right. FIG. 7C is a graphand bar chart showing the inhibition of 5α-Reductase Type 2 enzyme forthe supercritical CO₂ extract product as compared with Permixon®. Theseand other studies lead to the conclusion that a greater effect will beenabled from the enhanced LSESr, such as Experiments 5 and 6 (FIG. 4A),and will restore hair growth even more in AGA mouse models to block DHTeffects to an even greater degree than conventional hexane extracts orconventional CO₂ extracted saw palmetto extracts.

The hair follicle includes lipids and especially fatty acids and freefatty acids. The highest concentration of lipids in human hair is about4.3 mg/g of free fatty acids. For example, the medulla of the hairfollicle includes squalene, triglycerides, cholesterol, wax esters, andfree fatty acids such as oleic acid and palmitic acid. The cortex of thehair follicle includes integral fatty acids such as linoleic acid andalpha-linoleic acid and non-covalently attached fatty acids,triglycerides, cholesterol, wax esters, and squalene. The interfacebetween the cortex and cuticle includes a lipid containingN-acetylglucosamine. The entire cuticle contains ceramides andcholesterol. An epicuticle includes 18-methyl eicosanoic acid (18-MEA)and an exocuticle includes free fatty acids and covalently-attachedfatty acids. An endocuticle includes free fatty acids andcovalently-attached fatty acids. Free fatty acids are common lipids inhair, mainly in the hair matrix cells.

Recent studies show that linoleic acid promotes hair growth in normalmice, and promotes the Anagen phase in normal shaved mice byup-regulating hair growth related proteins. After about 20 days,linoleic acid operated better than controls and equivalent to Minoxidilwith about 100% hair growth.

Referring now to FIG. 7D, there is illustrated a bar chart showing thedistribution of oral alpha-linolenic acid in guinea pigs. Linolenic acidwas mainly distributed in the fur and skin at about 46% total, of which54% was found in fur and 46% in skin. Over 70% of that lipid was foundin the free fatty acid form. There were 16 times more linolenic acid inthe fur and skin of the head than in the fur and skin of the rest of thebody. The authors of the study by Fu et al. entitled, “Novel Pathway ofMetabolism of α-Linolenic Acid in the Guinea Pig,” Pediatric Research,47, 414-417 (2000), assumed that linolenic acid was being secreted bythe sebaceous glands in the skin onto the fur. However, the authors donot test this assumption. This study stresses that the preprocessing andCO₂ extraction parameters that enhance linoleic and linolenic free fattyacids and include the higher ratio of the four bioactive free fattyacids would be beneficial over conventional hexane or CO₂ extracts.

It is known that oral free fatty acids may be localized to the skin andhair follicles in addition to the prostate where the fivealpha-reductase enzymes are located. The human hair contains a highpercentage of fatty acids, mainly in the free fatty acid form. Thus, itis possible that some of the linolenic acid may have instead becomeincorporated into the fur of the guinea pigs. The enhanced benefit ofthe free fatty acid linolenic acid for hair health and hair growth isevident.

The enhanced LSESr of the current invention may also be used to reduceacne since there is localization of 5α-Reductase enzyme in inflammatoryacne lesions and inhibition of 5α-Reductase has been shown to improveacne in men with androgenic hair loss. 5α-Reductase Type 1 enzymes arethe predominan isotype in acne lesions and are localized in thesebaceous glands. 5α-Reductase Type 2 enzymes are the minor isotype inacne lesions and are localized to sebaceous duct and endothelial cells.

The enhanced LSESr of the current invention inhibits 5α-Reductase 1 and2 enzymes that play an important role in androgenic hair thinning andeventually hair loss. The enhanced LSESr of the current inventioninterferes with DHT production in the scalp. The DHT impacts the hairgrowth cycle in genetically susceptible individuals, causing the hairfollicle to miniaturize, which results in hair thinning and eventuallyhair loss. Thus, the enhanced LSESr of the current invention inhibits5α-Reductase 1 and 2 enzymes that convert testosterone to DHT, and thus,supports a healthy hair growth cycle, hair health, and becomes animportant active ingredient to be used for supplements that help inhibitthinning hair.

The enhanced LSESr of the current invention has a high ratio of freefatty acids to total fatty acids that is greater than about 80.0% andenrichment of lauric, myristic, oleic and linoleic free fatty acidsgreater than about 82.0%, and inhibits 5α-Reductase types 1 and 2enzymes that play an important role in androgenic hair thinning andeventually hair loss. The enhanced LSESr of the current inventioninterferes with DHT production and reduces the amount of DHT that mayaccumulate in the scalp, and which impacts the hair growth cycle. DHTmay accumulate in the scalp and impact the hair growth cycle, causingthe hair follicle to miniaturize in genetically suscepti ble indivudalsand result in hair thinning and eventually hair loss. The enhanced LSESrof the current invention inhibits 5α-Reductase type 1 and type 2 enzymesthat convert testosterone to DHT and supports a healthy hair growthcycle and hair health. The enhanced LSESr may be an important activeingredient added to dietary supplements for addressing thinning hair andhelping to make hair thicker. The enhanced LSESr of the currentinvention has a higher ratio of free fatty acids to total fatty acids asshown by experiments and will support hair thickness and hair fullness,and operate as a natural and safe ingredient for hair health.

It is possible to add vitamin D with the enhanced LSESr of the currentinvention. The vitamin D receptor expressed in dermal papilla cells willmaintain hair follicle homeostasis, and especially anagen initiation.The dermal papilla cells are an important site for DHT impact on hair.The vitamin D receptor in both serums and tissues are significantlydifferent in adults with androgenic hair loss than other controls (p=0).An insufficient amount of vitamin D may mean that keratinocytes in hairfollicles are not able to regulate hair growth and shedding. It is knownthat women with mild-to-moderate female pattern hair loss (FPHL) havesignificantly higher mean serum vitamin D levels than women with severehair loss. The micro-inflammation that does not lead to scarring of thescalp has been evident in women with FPHL and may be related to the hairfollicle miniaturization process.

There are also synergies using the enhanced LSESr of the currentinvention with zinc because it has been shown to increase the proportionof follicles in anagen, hair volume, and hair appearance in men withAGA. Zinc deficiency may reduce the efficacy of Minoxidil treatment inmen with AGA. Zinc may inhibit DHT production by limiting production ofNADPH, which is necessary for the function of 5α-Reductase enzymes. Acombination topical lotion containing the enhanced LSESr of the currentinvention and an herbal supplement containing zinc may be 50% moreeffective than either alone in male and female AGA. Some studies haveshown that treatment with a topical saw palmetto combination product forfour weeks increased the average hair count and terminal hair count at12 and 24 weeks in 50 men with androgenic alopecia. The enhanced LSESrof the current invention will provide greater enhancements.

It also possible to formulate the enhanced LSESr of the currentinvention with rosemary oil, and optionally in combination with 5%Minoxidil. This combination should be significantly better thanMinoxidil alone and improve the mean hair diameter and hair mass. Theenhanced LSESr of the current invention may be combined with one or moreof vitamin D, zinc and rosemary oil to address androgenic-mediated hairthinning and loss, support hair growth and regrowth, and addressmicro-inflammation in the hair follicle in adults suffering fromandrogen-mediated hair loss. Zinc, which inhibits 5α-Reductase through adifferent mechanism of action than LSESr, may also act synergisticallywith the enhanced LSESr of the current invention to support hairfollicle health, growth of new hair, hair volume and fullness, andoverall hair health and appearance. The rosemary oil may actsynergistically to support optimal hair growth in patients already usingMinoxidil.

The enhanced LSESr of the current invention is produced usingpre-extraction processing parameters for saw palmetto berries andspecific processing parameters of ultra-high-pressure, supercritical CO₂extraction, which creates a hexane-like extract but without the harshorganic chemicals. It is rich in the free fatty acids which have goodbioavailability and are absorbed twice as well as triglycerides, andincludes better localization to the site of activity, such as the scalpor prostate as compared to triglycerides.

There now follows a description of parameters associated with thepreprocessing of ripe saw palmetto berries as pre-extraction parameters,which in combination with specific processing parameters in thesupercritical CO₂ extraction, will produce the enhanced LSESr as inExperiments 5 and 6 in FIG. 4A. A regression model may be described onthe reaction and interactions of the pre-extraction process for totalfree fatty acids, followed by a description of the supercritical CO₂extraction process used in conjunction with the saw palmetto berryprocessing to produce the enhanced LSESr. Together the pre-extractionprocessing and processing parameters of the CO₂ extraction, such asdescribed in the flowchart of FIG. 55 , will obtain the beneficial ratioof free fatty acids to total fatty acids as in Experiments 5 and 6 (FIG.4A), Lot Nos. 211022 and 211105. Free fatty acid consistency andmaximization is achieved by preprocessing the ripe saw palmetto berriesbefore the supercritical carbon dioxide extraction. A regression modelmay be established based on the reaction and interactions of the processto the total free fatty acids.

In an example, dried mature saw palmetto berries of less than about 12%to about 13% moisture, and preferably ranging from 10% to 12% moisture,were released for the production process by a batch reference number.The saw palmetto berries by batch were introduced to a cryogenic millingsystem to create a consistent fine powder. The cryogenic milling systemincluded a control feeder, and in an example, a 50 horsepower choppermill with ½ inch screen openings. Different horsepower ratings may beused depending on the type of machine. A cryogenic conveyor included aliquid nitrogen addition to create chopped berries at about -40° C. Inan example, the temperature was no greater than -20° C. This range canvary as much as 10-20 degrees (F or C) above and below the -40° C.value.

Frozen material was dropped by gravity into a 50 horsepower pulverizingmill with a 3/16 inch screen opening. The feed rate was typically about119+/-6 kilograms per hour (kg/hr) with a resulting mill amperage ofabout 4 amps, and an amperage range preferably of about 2.5 to 5.5 amps.The 50 horsepower chopper amperage had a similar amperage range and isin-line with the process at a consistent feed rate. The liquid nitrogenrequirements were about 20+/-1 standard cubic feet (SCF) cryogenicnitrogen per kilogram of chopped and dried saw palmetto berries toachieve a recommended process temperature of feed into the mill on acontinuous basis. This can vary by about 10% to 20% above and belowthese values.

The fine, milled saw palmetto powder about 100% less than about 4 meshand about 95% less than about 12 mesh was fed into super sacks at about600 to about 900 kilograms each on pallets. The entire lots oncecompleted were stored by lot in a warehouse under hold, until aged to atarget time. The aging was greater than about 15 days, and maypreferably about 15 days to about 30 days, and in another example, begreater than 20 days, but not excessive such as beyond 40 day aging, andin an example, about 15 days to about 30 days for best results. Thecryogenic milling followed by at least 15 days aging was found importantwith the CO₂ extraction parameters. The completed milled and aged sawpalmetto powder by lot was released to an extraction and separationprocess.

Referring now to FIG. 8 , there are shown a graph and chart of thecryogenic mill AC amperage used for the saw palmetto production, andshowing a histogram of the density versus the AC current amperage. Thedensity versus AC current curve is the amperage of the primary cryogenicmill in operation on frozen, chopped saw palmetto berries. A smalleroccurrence of amperage greater than about 5.5 may be associated with astart-up of short duration, but it is not as important to productquality since 100% of material passes through a screen. Mill loading maybe about 262.4 +/-13.2 pounds per hour with a 50 horsepower motor orabout 119 +/-6 kilograms per hour with a 37.3 kilowatt (kw) motor. Thechopper processing with ambient temperature saw palmetto berries has asimilar loading rate on a 50 horsepower motor.

The aging impact on the free fatty acids (FFA) with fine milled berriesas processed above was determined at a central Florida location havingambient warehouse conditions. An average temperature is shown in FIG. 9Afor December through June, reflecting a typical average year for thattime period, and showing the normal test temperature and the percentversus the temperature Fahrenheit. The graph in FIG. 9B shows the normaltest relative humidity percent on the horizontal line and percentage onthe vertical line.

Ambient aging conditions have an effect on the saw palmetto berries.Thus, various warehouse ambient conditions are important for the milledsaw palmetto, and aging is appropriate prior to extraction. FIG. 10A isa table showing various months and the temperature in degreesFahrenheit, and the months from December (12) to June (6) as an averagecycle. The table shows the mean, standard error mean, standarddeviation, the C variation, the median, the total remuneration (TR) forthe mean, the minimum, the maximum, the range, and N as the populationsize. The temperature may range in this example from about 59° F. to 79°F. and in a wider range, about a minimum of 40° F. to maximum of about87° F., and a mean of about 59° F. to about 78° for best results. FIG.10B is a similar table as in FIG. 10A, but showing the percentage ofrelative humidity and showing a range from about 30% to about 97%, and amean of about 67% to about 78% for best results.

The mature, dried, and milled saw palmetto berries that are aged arethen extracted with the enhanced processing parameters. The scatter plotgraph data shown in FIG. 11 represents the data for over 60 campaigns of1200 MT dried berries or over 9 MM pounds of harvested berries. Freefatty acids versus fatty acids for the five actives of lauric, myristic,oleic, linoleic, and linolenic acids are shown as the scatter plot inFIG. 11 showing on the vertical axis the five actives FFA/FA versus themill and hold time in days on the horizontal, and showing the relativeflat line curve at about 20-35 days and the peak at about 28 days. Agingshould occur at greater than 15 days, but it is evident that beyond 30days in this example, the results and effectiveness were decreasedslightly.

Referring now to FIGS. 12A and 12B, the statistics for the active fivefatty acids and the active five free fatty acids that result from theprocessing of the dried saw palmetto berries relative to starting berrymoisture are shown. In FIG. 12A, the bar chart for the key fatty acidsand five active free fatty acids are shown when the starting berrymoisture is below the desired specifications corresponding to less than10% moisture. This bar chart also shows the graph line for the averageof the five actives and ratio FFA/FA. In FIG. 12B, the bar chart showsthe average of the five key fatty acids and free fatty acids similar toFIG. 12A when the starting berry moisture is in specification of about10% to 12% moisture and showing the graph line similar to that shown inFIG. 12A for the average of five actives FFA/FA.

Referring now to FIG. 13 , a series of graphs as scatter plots are shownthat relate to the process conditions and parameters to resolve qualityfor the percentage of free fatty acids relative to fatty acids versusthe time between harvest and extraction. The scatter plots areillustrated for the five active free fatty acids relative to fatty acidsversus time between harvest and extraction. The graphs on the left showan average hold time between initial harvest and extraction via thesupercritical CO₂ extraction of about 200 days. This includes thepost-mill hold time. Thus berries are selected and dried, then groundand held for at least 15 days.

In FIG. 14 , a line graph is shown for the analysis of the variance(ANOVA) for the percentage of free fatty acids/fatty acids on the fivekey fatty acids in the saw palmetto per the mill and hold time andshowing an alpha (a) of 0.05. The mean is shown on the vertical axis andbrackets hold time on the horizontal axis. Less than 10 days isstatistically significant with 95% less than average. A greater than 15days hold time is statistically improved with a 95% confidence versusall hold times less than 15 days.

The hold time impact on the variance of the percentage of free fattyacids and the fatty acids are shown in the test results as a bracketedline of FIG. 15 , showing a test for equal variances for the fiveactives of free fatty acids/fatty acids versus the brackets hold time.There are multiple intervals for the standard deviation of alpha (a)equal to 0.05. The longer hold time greater than 10 days isstatistically improved at a 95% confidence on the variance versus lessthan 10 days. The analysis of variance (ANOVA) for the specific fiveactive fatty acids as lauric acid, myristic acid, oleic acid, linoleicacid, and linolenic acid are shown in the line graphs of FIGS. 16-20respectively, and showing the brackets hold time in the horizontal axisand the mean in the vertical axis. In each graph, the specific acid isidentified as a percentage of free fatty acid/fatty acid in the sawpalmetto per mill and hold with alpha (α) = 0.05. The graph lines showthe benefits of milling and holding as described above and with thebetter results at about 15 days and greater.

Different fatty acid statistical tables with ranges are shown in FIGS.21-26 . In FIG. 21 , the statistics for the five active fatty acidsidentified above for prostate health are shown, followed by thestatistical tables for lauric acid (FIG. 21 ), myristic acid (FIG. 22 ),oleic acid (FIG. 23 ), linoleic acid (FIG. 24 ), and linoleic acid (FIG.26 ). The post mill hold time in days is illustrated with the values asin the tables of FIGS. 10A and 10B. The benefits of the longer hold timeare evident with both the mean and median have the better values for allexamples above 15 days. For example, in FIG. 21 , the highest percentageof FFA/FA is 87.80% at greater than 15 days, post-hold time, which wasused to obtain the advantageous results for the enhanced LSESr such asshown with Experiments 5 and 6 of FIG. 4A, giving the four major activeFFA’s greater than 82.0%, and close to that with Experiment 4.

This evidence also shows that the saw palmetto berries that areharvested and dried have various levels of fatty acids in a specificrelationship to each other. The free fatty acid quality for the samefatty acids varies naturally over the season and region of harvest. Byselecting specific berry preprocessing conditions and parameters andspecific supercritical CO₂ extraction parameters, it is possible tomaximize and standardize to a tight variance the individual free fattyacid levels and their ratio relative to fatty acids that are importantfor prostate health and hair health. These results also add insight intothe interactions and reactions occurring with the fatty acids and freefatty acids as based on the processing parameters and the post mill holdtime.

FIG. 27 is a graph showing the percentage of the actives for the freefatty acids/fatty acids versus the post mill hold time of the fiveactive fatty acids as the lauric, myristic, oleic, linoleic, andlinolenic fatty acids where alpha (a) = 0.05, and showing the benefitsof hold times at about 15 days compared to a lesser hold time. The testfor equal variances for the five actives as free fatty acids/fatty acidsversus the brackets hold time is shown in the bracket line graph of FIG.28 where the lower standard deviation bracket is better. When thebrackets do not overlap with other conditions than statisticallydifferent at 95% confidence, it is evident that the longer hold time ofpost-milling of about 15 days is better. There are multiple comparisonintervals for the standard deviation or alpha (a) when it is equal to0.05. Multiple comparisons are shown where the p-value is equal to 0.024and Leverne’s test has a p-value of 0.005.

Referring now to FIGS. 29-34 , the different interactions and reactionsthat occur based on processing conditions and notably the post mill holdtime are shown with the five actives starting with lauric acid in FIG.29 , followed by myristic acid (FIG. 30 ), oleic acid (FIG. 31 ),linoleic acid (FIG. 32 ), and linolenic acid (FIG. 33 ). The total fattyacids weight fraction versus the post mill hold time is shown in theline graph of FIG. 34 . In all graphs, alpha (a) = 0.05 and bracketshold time is on the horizontal axis and the mean on the vertical axis.The lauric fatty acid may be statistically higher on average when thehold time is short (FIG. 29 ). The myristic acid weight fraction isshown in FIG. 30 and it is not evident that the myristic fatty acid isdifferent on average across time.

The oleic acid weight fraction is shown in FIG. 31 and the oleic fattyacid increases directionally and then standardizes over the hold time.The linoleic acid weight fraction versus the post milling hold time isshown in FIG. 32 and it is not evident that the linoleic fatty acid isdifferent on average over the hold time, but the direction pattern isevident on average and then stabilizes.

The linolenic acid weight fraction versus the post milling hold time isshown in FIG. 33 and it is not evident that the linolenic fatty acid isdifferent on the average over the hold time, but the direction patternis strongly evident on average and then stabilizes. The total fattyacids weight fraction versus the post mill hold time is shown in FIG. 34and from that and other graphs. The data from preceding graphs andcharts display generally that the preprocessing parameters aid inobtaining the enhanced LSESr to obtain the higher percentage of fouractive free fatty acids (lauric, myristic, oleic and linoleic) whenthere are specific milling parameters and post-mill aging greater than15 days.

There are probable interactions as one of the active fatty acids shift.An analysis is helpful about which fatty acid shifts the most and whichfatty acids are important to the total free fatty acids and total fattyacids. The data may be analyzed by doing a regression analysis todetermine which fatty acids and free fatty acids interact the most tothe total free fatty acid in the process with the saw palmetto extract.

The table in FIG. 35 shows a multiple regression for the total freefatty acids versus the fatty acids and showing a model equations reportwith the four active free fatty acids myristic X1, oleic X2, linoleicX4, linolenic X4, and X5 as bracket hold time in days. The equation usesmass fractions for the individual free fatty acids, and the hold time isdays after milling. Lauric acid did not show up as a major contributorthat accounts for variance, and thus, it was dropped from the model andin FIG. 35 , it is not shown. Myristic, oleic, linoleic and linolenicare illustrated. Lauric acid is considered important to the total freefatty acids, however. It is evident that there are interactions betweenfree fatty acids and the higher order activity on some free fatty acids.The sign of the terms are important since interactions are reducing andothers are increasing the total free fatty acid levels. This can beexpected as the reactions are occurring during the process.

Referring now to the bar charts in FIG. 36 , a multiple regression fortotal free fatty acids/fatty acids is shown with a model buildingreport. The bar charts show the model building sequence and theincremental impact of x variables and each x regressed on all otherterms. The model building sequence on the left displays the order inwhich the items were added or removed. The bar chart on the upper rightshows the incremental impact of oleic acid as compared to linolenic acidas the smallest increase for the R-squared percentage.

The graphs in FIG. 37 show the interactions effect for free fatty acids,where only significant effects are shown. As illustrated, the multipleregressions for the total free fatty acids/fatty acids effect report areshown. The interaction plots show how the total free fatty acids/fattyacids changes if you change the settings of two “X” variables. The maineffects plots for total free fatty acids/fatty acids are shown withchanges in an “X” variable. The power and significance are highlysignificant with the low P-value and very high R² as shown in FIG. 38 ,which shows a summary report for multiple regressions for the total freefatty acids/fatty acids. The model may be diagnosed with the multipleregression shown in FIG. 39 . There is a notable similarity in the shorttime from harvest to extraction and short hold time post milling.

The multiple regression for the total free fatty acids and modelbuilding report are shown. The variables for maximizing total free fattyacids and the reactions and interaction of the fatty acids and freefatty acids are shown as they contribute to total free fatty acid andcan be analyzed to determine any maximizing behavior. The five activefatty acids are grouped together as one factor (with free fatty acids aslauric, myristic, oleic, linoleic, and linolenic) and the volatile shortchain fatty acids then analyzed. The R² adjusted is 99.96% and theP-value is about 0.006 on the regression, which are very good values.The interactions and second order reactions with the shorter chainsoperating towards maximizing the total free acid.

The multiple regression for free fatty acids is shown in FIG. 40 . Allfive factors contribute to the model and do not correlate with oneanother. A highly significant relationship occurs between the X factorsand the percentage of total fatty acids, with a P-value less than about0.001. With the R² adjusted at 99.96%, the model of factors account for99.96% of the variations seen in the saw palmetto extract for total freefatty acids. The summary of the results are shown in the chart andgraphs of FIGS. 41-43 and the symmetry of the model is good, so thereare no special causes in the data, which is accepted. This data pointsto the acceptable conclusion that the preprocessing of berries in aspecific manner before CO₂ extraction has a significant effect on thefinal LSESr.

The results of the various experiments, such as described relative tothe data of FIGS. 4A, 4B, 5 and 6 and the statistical analysis of thevarious graphs and charts described above, indicate that the key fattyacids as lauric, myristic, oleic, and linoleic free fatty acids areimportant. It is possible to have lower total fatty acids of greatertotal and free fatty acids, but the more important aspect is how much ofthe free fatty acid percentage is contributed by the important fouractive free fatty acids. The data also reinforces that the preprocessingof the saw palmetto berries is important. A typical hops styleextraction can have low free fatty acids in some cases.

The fitted line plot of FIG. 44 shows a relationship between the lauricacid percentage weight/weight and total percentage of free fatty acids.There is variability in the data that would make the difference betweenabout 75% to about 77% insignificant. Directionally, it is known thatgreen berry oil has higher lauric acid versus ripe mature berries. Withthe mature berries and the aging, the lauric acid is lower in terms ofmaturity, but the free fatty acids are higher and more consistent aroundan average. This example applies especially compared to green berry oil.

Another scatter plot of the lauric acid and free fatty acid ratio versusthe calculated percentage of oil in berries is shown in the graph ofFIG. 45 . A goal was to maximize the percentage of free fatty acids andthe total oil content for extraction. The lower ratio values are betterfor saw palmetto oil effectiveness. Similar results are shown in thescatter plot graphs of FIGS. 46, 47, and 48 . The hold time in days ofFIG. 47 illustrates the lower ratio and tighter consistency with agepost-milling and showing an aging of around 25 to 26 days as effective,and thus, greater than 15 days and preferably about 15 to 30 days agingas post-milling. In one aspect, a goal is to age post-milling to 20 daysor later for full aging. As shown in FIG. 48 , the finished oil for thebiomass of around 12.6% to 12.7% is effective.

Referring now to FIGS. 49-52 , an analysis of variance for free lauricacid per mill hold time in FIG. 49 is shown with the analysis ofvariance for free fatty acids for the mill and hold time shown in FIG.50 , and the test for equal variances shown for lauric acid (FIG. 51 )and the total free fatty acids (FIG. 52 ). The mean value of free lauricacid is significantly improved and higher when aging is greater than 15days and shows a 95% confidence in the data as shown in FIG. 49 . Thedata is statistically and significantly lower when less than 10 days(FIG. 49 ). The mean value of total free fatty acids is significantlyimproved and higher when aging is greater than 15 days and showing a 95%confidence (FIG. 50 ) and significantly and statistically lower whenless than 10 days. As shown in FIG. 51 , the standard deviation of freelauric acid is improved as a result of the aging process, such asgreater than 15 days, and there is a significantly statisticalimprovement in the standard deviation of the percentage of free fattyacids when aging is greater than 15 days and showing a 95% confidence.

Referring now to FIG. 53 , there is illustrated an analysis of varianceof free fatty acids per mill and hold for a harvest season and showingthe mean by bracketed for post-cryomilling and aging. The dots in thisgraph and other similar graphs as described before represent the meanvalue for these conditions. The first line labeled A is the overall meanvalue for the data set. The lines labeled B show the 95% confidencelines as function of product variance and sample size. The dots labeledC indicate the mean value is 95% confident as a different higher orlower, and in this specific case, when the post-mill aging is greaterthan 15 days, the mean value for percentage of free fatty acids is 95%confidently higher than the lower aging times.

Using the same data as above, the test for equal variance (FIG. 54 )suggests that the estimate range for the standard deviation across agedtime brackets is different and lower than 95% confidence level when theaging is greater than 10 days. Visually, when the brackets in the chargedo not overlap, then it is possible to state that it significantlydifferent with an alpha of equal to about 0.05. The practicalimplication is that the variance of the finished product will besignificantly reduced when the percentage of free fatty acids ismaximized, thus, creating a high quality extract.

Referring now to FIG. 55 , there is illustrated a high level flowchartgenerally at 500 showing the sequence used to manufacture thesupercritical CO₂ enhanced LSESr as in Experiments 5 and 6 of FIG. 4A.These examples of the enhanced LSESr have a high ratio of free fattyacids to total fatty acids of greater than about 80.0% and maximizingthe four bioactive free fatty acids described above. This better profileis driven by the pre-extraction handling of the ripe berries asdescribed above and the supercritical CO₂ extraction parameters asexplained in the flowchart of FIG. 55 and relative to the flowchart ofFIG. 1 . The total fatty acids is less important than the ratio of freeversus total fatty acids in order to optimize the contribution of thefour major bioactive fatty acids as a percentage of total fatty acids,with the four bioactive fatty acids in this case identified as lauric,myristic, oleic, and linoleic. For hair growth, linolenic free fattyacid also becomes an important contributor.

In this example, the process starts (Block 502) where 19,958 kilogram(kg) of dried saw palmetto (Block 504) was fed through a feed hopper(Block 506). A magnet extracted any magnetic particles or contaminants(Block 508). The product was fed into the hammer mill (Block 510) whereliquid nitrogen (Block 512) was applied at about -40° C. to about -30°C., which could possibly range from about -44° C. to about -27° C., avariation of about 10%. The product was aged (Block 514) as noted above,up to about 15 days at least, and preferably at least greater than 15-20days and up to about 15 to 30 days, or even 15 to 40 days. Thetemperature may vary during ageing as noted above with a desired mean ofabout 59° F. to about 78° F. As noted in the data above, aging togreater than 15 days has been found important together with the uniqueCO₂ extraction parameters to optimize the contribution of the fourbioactive free fatty acids.

Reference is made to some of the aging parameters described above thatwere followed. After aging, the aged and milled dried saw palmettopowder was placed into the supercritical CO₂ extractor and carbondioxide used as the supercritical fluid (Block 516) was applied at about550 bar at 85° C. for CO₂ extraction (Block 518). These values can rangefrom about 495 bar to about 605 bar and 77° C. to about 94° C. In thisexample, based upon the 19958 kg initial amount, the spent marc for thesolid waste leftover (Block 520) was obtained at about 16,645 kg.

The first fractionation (Block 522) occurred and the extract wasseparated at a first stage and the amount as the first separated extract(Block 524) was about 2,426 kg. This first fractionation occurred atabout 140 bar at about 77° C. in this example. This pressure could rangefrom about 126 bar to about 350 bar, and the temperature range fromabout 35° C. to about 85° C. A second fractionation (Block 526) occurredat about 55 bar and at about 25° C. and the second separated extract(Block 528) was about 1,164 kg. The pressure range may be about 30 barto about 80 bar, and a temperature range may be about 12° C. to about28° C. The first and second separated extracts may be dewatered anddecanted (Block 530), followed by blending to form a final product ofabout 2,377 kg (Block 532) corresponding to the enhanced LSESr. Theprocess ends (Block 534).

FIG. 56 is a table of comparative data for high and low pressureextraction (250 bar at 70° C.entigrade vs. 500 bar at 85 degreesCentigrade) of two lot numbers using the same raw material such asdescribed above. There are differences in concentrations of total freefatty acids, but the proportion of lauric, myristic, oleic, and linoleicto all free fatty acids is the same (81%) between the extractionconditions since the fractionation conditions were similar. This tableshows support to the inventive fractionation requirement described aboverelative to the flowcharts of FIGS. 1 and 55 .

This application is related to copending patent applications entitled,“COMPOSITION AND METHOD HAVING ENHANCED LSESr TO MAINTAIN AND PROMOTEURINARY AND PROSTATE FUNCTION IN A HUMAN,” and “COMPOSITION AND METHODHAVING ENHANCED LSESr TO MAINTAIN AND PROMOTE HAIR HEALTH AND GROWTH,”which are filed on the same date and by the same assignee and inventors,the disclosures which are hereby incorporated by reference.

Many modifications and other embodiments of the invention will come tothe mind of one skilled in the art having the benefit of the teachingspresented in the foregoing descriptions and the associated drawings.Therefore, it is to be understood that the invention is not to belimited to the specific embodiments disclosed, and that themodifications and embodiments are intended to be included within thescope of the dependent claims.

1. A method of producing an enhanced lipidosterolic extract of Serenoarepens (LSESr), comprising: cryogenically milling at a temperature nogreater than -20° C.entigrade dried, saw palmetto berries having greaterthan about 10% moisture into a saw palmetto powder; ageing the sawpalmetto powder for at least 15 days; subjecting the aged saw palmettopowder to supercritical CO₂ extraction at a first extraction pressureand first extraction temperature to produce a CO₂ extract; fractionatingthe CO₂ extract at a first separation pressure and first separationtemperature to produce a first fraction, followed by fractionating at asecond separation pressure and second separation temperature to producea second fraction and an enhanced LSESr having a ratio of free fattyacids to total fatty acids that is greater than about 80.0% and anenrichment of lauric, myristic, oleic and linoleic acids as free fattyacids to total free fatty acids that is greater than about 82.0%.
 2. Themethod of claim 1 wherein the enhanced LSESr has a balance of about90.0% of total fatty acids to about 72.0% of free fatty acids.
 3. Themethod of claim 1 wherein the enhanced LSESr has an enrichment oflauric, myristic, oleic and linoleic free fatty acids to total freefatty acids of about 82.0% to about 84.0%.
 4. The method of claim 1wherein the enhanced LSESr has a ratio of free fatty acids to totalfatty acids of about 80.0% to about 82.0%.
 5. The method of claim 1wherein the supercritical CO₂ extraction occurs at an extractionpressure of about 495 bar to about 695 bar at a temperature of about 77to about 94° Cntigrade.
 6. The method of claim 1 wherein the firstfractionation occurs at about 126 bar to about 350 bar at a temperatureof about 35 to about 85° Cntigrade.
 7. The method of claim 1 wherein thesecond fractionation occurs at about 30 bar to about 80 bar at atemperature of about 12 to about 28° Cntigrade.
 8. The method of claim 1comprising cryogenically milling the saw palmetto berries into a groundsaw palmetto powder having about 100% less than about 4 mesh and about95% less than about 12 mesh.
 9. The method of claim 1 comprisingcryogenically milling at a temperature of about -50 to about -30°Cntigrade.
 10. The method of claim 1 wherein the enhanced LSESr has aperoxide value that is less than about 3 meq/kg and shelf stability ofat least about 4 years without added antioxidants and stabilizers. 11.The method of claim 1 comprising ageing the saw palmetto powder forabout 15 to about 30 days.
 12. The method of claim 1 comprising ageingthe saw palmetto powder at a temperature from a mean of about 59 degreesto about 78° F.
 13. A method of producing an enhanced lipidosterolicextract of Serenoa repens (LSESr), comprising: cryogenically milling ata temperature from about -50 to about -30° C.entigrade dried, sawpalmetto berries having about 10% to about 13% moisture into a sawpalmetto powder having a fineness where about 100% is less than about 4mesh and about 95% is less than about 12 mesh; ageing the saw palmettopowder for at least 15 days at a temperature from a mean of about 59 toabout 78° F.; subjecting the aged saw palmetto powder to supercriticalCO₂ extraction at an extraction pressure of about 495 bar to about 695bar at a temperature of about 77 to about 94° C.entigrade to produce aCO₂ extract; fractionating the CO₂ extract at a first separationpressure of about 126 bar to about 350 bar at a first separationtemperature of about 35 to about 85 degrees Centigrade to produce afirst fraction, followed by fractionating at about 30 bar to about 80bar at a temperature of about 12 to about 28° C.entigrade to produce asecond fraction, and blending first and second fractions to produce anenhanced LSESr having an enrichment of lauric, myristic, oleic andlinoleic free fatty acids to total free fatty acids of about 82.0% toabout 84.0%, and a ratio of free fatty acids to total fatty acids ofabout 80.0% to about 82.0%.
 14. The method of claim 13 wherein theenhanced LSESr has a peroxide value that is less than about 3 meq/kg andshelf stability of at least about 4 years without added antioxidants andstabilizers.
 15. The method of claim 13 comprising ageing the sawpalmetto powder for about 15 to about 30 days.
 16. A dietary supplementcomposition, comprising a shelf stable, supercritical CO₂ fluidextracted, enhanced lipidosterolic extract of Serenoa repens (LSESr),the LSESr comprising: an enrichment of lauric, myristic, oleic andlinoleic free fatty acids to total free fatty acids greater than about82.0%; and a ratio of free fatty acids to total fatty acids greater thanabout 80.0%.
 17. The dietary supplement composition of claim 16 whereinthe enhanced LSESr has a peroxide value that is less than about 3 meq/kgand shelf stability of at least about 4 years without added antioxidantsand stabilizers.
 18. The dietary supplement composition of claim 16wherein the composition is formulated into an oral dosage form.
 19. Thedietary supplement composition of claim 16 wherein the composition isformulated into an oral dosage form and in a capsule of about 160 mgb.i.d. to about 320 mg, or about minimum 200 mg per dose of enhancedLSESr.
 20. The dietary supplement composition of claim 16 wherein thecomposition includes less than about 0.2 percent w/w of saw palmettosterols.
 21. The dietary supplement composition of claim 16 wherein thecomposition includes a dry excipient comprising at least one of silicondioxide, calcium silicate, calcium phosphate, magnesium oxide, magnesiumcarbonate, calcium carbonate, rice fiber and maltodextrin.
 22. Thedietary supplement composition of claim 16 wherein the enhanced LSESrhas a balance of about 90.0% of total fatty acids to about 72.0% of freefatty acids.
 23. The dietary supplement composition of claim 16 whereinthe enhanced LSESr has an enrichment of lauric, myristic, oleic andlinoleic free fatty acids to total free fatty acids of about 82.0% toabout 84.0%.
 24. The dietary supplement composition of claim 16 whereinthe enhanced LSESr has a ratio of free fatty acids to total fatty acidsof about 80.0% to about 82.0%.